BIOLOGY 

LIBRARY 

G 


THE  PRINCIPLES 


BACTERIOLOGY 


A  PRACTICAL  MANUAL  FOR  STUDENTS 
AND  PHYSICIANS. 


BY 


A.   C.   ABBOTT,   M.D., 

FIRST   ASSISTANT,    LABORATORY    OF    HYGIENE,  UNIVERSITY    OF    PENNSYLVANIA, 
PHILADELPHIA. 


WITH      ILLUSTRATIONS. 


PHILADELPHIA: 

LEA  BROTHERS   &  CO. 

1892. 


ftS- 

BIOLOGY 

LIBRARY 

G 


Entered  according  to  Act  of  Congress,  in  the  year  1891,  by 

LEA  BROTHERS  &  CO., 
In  the  Office  of  the  Librarian  of  Congress  at  Washington,  D.  C. 


DORNAN,  PHINTEK. 


PREFACE. 


IN  preparing  this  book  the  author  has  kept  in  mind 
the  needs  of  the  student  and  practitioner  of  medicine, 
for  whom  the  importance  of  an  acquaintance  with  prac- 
tical bacteriology  cannot  be  overestimated. 

It  is  to  advances  made  through  bacteriological  re- 
search that  we  are  indebted  for  much  of  our  knowledge 
of  the  conditions  underlying  infection,  and  for  the  elu- 
cidation of  many  hitherto  obscure  problems  concerning 
the  etiology,  the  modes  of  transmission,  and  the  means 
of  prevention  of  infectious  maladies. 

Only  within  a  comparatively  short  time  have  students 
and  physicians  been  enabled  to  obtain  the  systematic 
instruction  in  this  science  that  is  of  value  in  aiding 
them  in  their  efforts  to  check  disease.  The  rapid  in- 
crease in  the  number  who  are  availing  themselves  of 
these  opportunities  speaks  directly  for  the  practical 
value  of  the  science. 

As  the  majority  of  those  undertaking  the  study  of 
bacteriology  do  so  with  the  view  of  utilizing  it  in 
medical  practice,  and  as  many  of  these  can  devote  to  it 
but  a  portion  of  their  time,  it  is  desirable  that  the 


IV  PREFACE. 

subject-matter  be  presented   in  as  direct  a  manner   a^ 
possible. 

Presuming  the  reader  to  be  unfamiliar  with  the  sub- 
ject, the  author  has  restricted  himself  to  those  funda- 
mental features  that  are  essential  to  its  understanding. 
The  object  has  been  to  present  the  important  ideas  and 
methods  as  concisely  as  is  compatible  with  clearness, 
and  at  the  same  time  to  accentuate  throughout  the 
underlying  principles  which  govern  the  work. 

With  the  view  of  inducing  independent  thought  on 
the  part  of  the  student,  and  of  diminishing  the  fre- 
quency of  that  oft-heard  query,  "What  shall  I  do 
next?"  exj>erirneuts  have  been  suggested  wherever  it 
is  possible.  These  have  been  arranged  to  illustrate  the 
salient  points  of  the  work  and  to  attract  attention  to  the 
minute  details,  upon  the  observation  of  which  so  much 
in  bacteriology  depends. 

A.  C.  A. 

PHILADELPHIA,  December,  1891. 


CONTENTS. 


INTRODUCTION. 

PAGE 

"  Omne  vivum  ex  vivo  " — The  overthrow  of  the  doctrine 
of  spontaneous  generation 13-21 

CHAPTER   I. 

Definition  of  bacteria — Their  place  in  nature — Differ- 
ence between  parasites  and  saprophytes — Nutrition  of 
bacteria — Products  of  bacteria — Their  relation  to  oxygen — 
Influence  of  temperature  upon  their  growth  .  .  .  22-28 

CHAPTER   II. 

Morphology  of  bacteria — Grouping — Mode  of  multipli- 
cation— Spore-formation — Motility 29-36 

CHAPTER    III. 

Principles  of  sterilization  by  heat — Different  methods 
employed — Principles  of  discontinued  sterilization — Ster- 
ilization under  pressure— Apparatus  employed  .  .  37-48 

CHAPTER   IV. 

Disinfection— Antiseptics — Inorganic  salts  as  disinfec- 
tants— The  value  of  corrosive  sublimate — Heat  .  .  49-53 

CHAPTER  V. 

The  principles  involved  in  the  methods  of  isolation  of 
bacteria  in  pure  culture  by  the  plate  method  of  Koch — 
Materials  employed 54-58 


VI  CONTENTS. 


CHAPTEK   V  I 

I'A«iK 

Preparation  of  media—  Bouillon,  gelatin,  agar-agar, 
potato,  blood-serum,  etc  ........  .")9-7(; 

CHAPTEK    VII. 

Preparation  of  the  tubes,  flasks,  etc.,  in  which  the  media 
are  to  be  preserved  ........  77-80 

CHAPTER   VIII. 

Technique  of  making  plates  —  Esmarch  tubes,  Petri 
plates,  etc.  .........  81-00 

CHAPTER    IX. 

The  incubator  used  in  bacteriological  work  —  (Jas-piv— 
ure  regulator  —  Thermo-regulator  —  The  form  of  burner 
employed  in  heating  the  incubator  .....  91-98 

CHAPTER   X. 

The  study  of  colonies—  Their  naked-eye  peculiarities 
and  their  appearance  under  different  conditions  —  Differ- 
ences in  the  structure  of  colonies  from  different  species  of 
bacteria  —  Stab  cultures  -Slant  cultures  ....  99-103 

CHAPTER   X  I 

Systematic  study  of  an  organism—  Steps  necessary  in 
identifying  an  organism  as  a  definite  species  .  .  .  104-121 

CHAPTER    XII. 

Methods  of  staining  —  Solutions  employed  —  Preparation 
and  staining  of  cover-slips—  Preparation  of  tissues  for 
section-cutting  —  Staining  of  tissues  —  Special  staining 
methods  ..........  122-150 

CHAPTER   XIII. 

Inoculation  of  animals  —  Subcutaneous  inoculation  — 
Intra-venous  injection  .......  151-158 


CONTENTS.  yii 

CHAPTER   XIV. 

PAGE 

Post-mortem  examination  of  animals— Bacteriological 
examination  of  the  tissues— Disposal  of  tissues  and  dis- 
infection of  instruments  after  the  examination  .  .  159-163 

CHAPTER   XV. 

Scheme  for  the  complete  study  of  an  organism      .         .164,  165 


PRACTICAL   APPLICATION   OF   THE 
METHODS  OF  BACTERIOLOGY. 

CHAPTER   XVI. 

To  obtain  material  upon  which  to  begin  work        .         .     167-170 

CHAPTER    XVII. 

Various  experiments  in  sterilization — Steam  and  hot-air 
methods  of  sterilizing 171-175 

CHAPTER   XVIII. 

Bacteriological  study  of  water,  air,  and  soil — Methods 
of  counting  the  colonies  on  the  plates — Wolff  hiigel's 
counting  apparatus— Sedgwick's  method  ....  176-191 

CHAPTER   XIX. 

Inoculation  experiments  with  sputum — Sputum  sep- 
ticaemia— Septicaemia  resulting  from  the  presence  of  the 
micrococcus  tetragenus  in  the  tissues — Tuberculosis  .  .  192-200 

CHAPTER   XX: 

Tuberculosis — Microscopic  appearance  of  miliary  tuber- 
cles—Diffuse caseation— Cavity-formation — Encapsulation 
of  tuberculous  foci — Primary  infection — Modes  of  infec- 
tion— Location  of  the  bacilli  in  the  tissues — Staining 
peculiarities  .........  201-216 


V1I1  CONTENTS. 

CHAPTER   XXI. 

I'AOK 

Suppuration — The  staphylococeus  pyogenes  aurous        .     217-224 

CHAPTER  XXII. 

Typlioid  fever — Study  of  the  organism  concerned  in  its 
production 225-231 

CHAPTER   XXIII. 

Study  of  the  bacillus  of  anthrax,  and  the  effects  pro- 
duced by  its  inoculation  into  animals— Peculiarities  of  the 
organism  under  varying  conditions  of  surroundings  .  232-242 

CHAPTER  XXI  V. 

Bacteriology  of  diphtheria — Behavior  of  the  bacillus 
diphtherias  in  the  tissues  of  susceptible  animals  .  .  243-252 

CHAPTER   XXV. 

Experiments  illustrating  precautions  to  be  taken  in  the 
study  of  disinfectants  and  antiseptics — Skin-disinfection  .  253-257 


BACTERIOLOGY. 


INTRODUCTION. 

"Oinne  vivum  ex  vivo" — The  overthrow  of  the  doctrine  of 
spontaneous  generation. 

THE  study  of  Bacteriology  may  be  said  to  have  had 
its  birth  with  the  observations  made  by  Antony  van 
Leeuwenhoek  in  the  year  1675.  Though  it  is  during 
the  past  deeade  and  a  half  that  this  line  of  research  has 
received  its  greatest  impulse,  yet  by  a  review  of  the 
developmental  stages  through  which  it  has  passed  in  its 
life  of  more  than  two  centuries,  we  see  that  it  has  a 
most  interesting  and  instructive  history.  From  the  very 
beginning  its  history  is  inseparably  connected  with  the 
history  of  medicine,  and  as  ft  now  stands  its  relations  to 
hygiene  and  preventive  medicine  are  of  the  most  im- 
portant nature.  It  is,  indeed,  to  a  more  intimate  ac- 
quaintance with  the  biological  activities  of  the  micro- 
organisms that  modern  hygiene  owes  much  of  its  value, 
and  our  knowledge  upon  infectious  diseases  has  been 
developed  to  the  position  it  now  occupies.  Though  the 
contributions  which  have  done  most  to  place  bacteriology 
on  the  footing  of  a  science  are  those  of  recent  years,  still, 
during  the  earlier  stages  of  its  development,  many  obser- 
vations were  made  which  formed  the  foundation  work 
for  much  that  was  to  follow.  Before  regularly  begin- 

2 


14  BACTERIOLOGY. 

ning  our  studies,  therefore,  it  may  be  of  advantage  to 
acquaint  ourselves  with  the  more  prominent  of  these 
investigations. 

Antony  van  Leeuwenhoek,  the  first  to  describe  tin- 
bodies  now  recognized  as  bacteria,  was  born  at  Delft, 
in  Holland,  in  1632.  He  was  not  considered  a  man  of 
liberal  education,  having  been  during  his  early  years  an 
apprentice  to  a  linendraper.  During  his  apprenticeship 
he  learned  the  art  of  lens-grinding,  in  which  he  became 
so  proficient  that  he  eventually  perfected  a  lens  by 
means  of  which  he  was  enabled  to  see  objects  of  much 
smaller  dimensions  than  any  hitherto  seen  with  the 
microscopes  in  existence  at  that  date.  At  the  time  of 
his  discoveries  he  was  following  the  trade  of  linendraper 
in  Amsterdam. 

In  1675  he  published  the  fact  that  he  had  succeeded 
in  perfecting  a  lens  by  means  of  which  he  could  detect 
in  a  drop  of  rain  water  living,  motile  animalcules  of  the 
most  minute  dimensions — smaller  than  anything  that 
had  hitherto  been  seen.  Encouraged  by  this  discovery, 
he  continued  to  examine  various  substances  for  the 
presence  of  what  he  considered  animal  life  in  its  most 
minute  form.  He  found  in  sea- water,  in  well-water,  in  the 
intestinal  canal  of  frogs  and  birds,  and  in  his  own  diar- 
rhoea! evacuations,  objects  that  differentiated  themselves 
the  one  from  the  other,  not  only  by  their  shape  and  size, 
but  also  by  the  peculiarity  of  movement  which  some  of 
them  were  seen  to  possess.  In  the  year  1683  he  dis- 
covered in  the  tartar  scraped  from  between  the  teetli 
a  form  of  microorganism  upon  which  he  laid  special 
stress.  This  observation  he  embodied  in  the  form-  of 
a  contribution  which  was  presented  to  the  Royal  So- 
ciety of  London  on  September  14,  1683.  This  paper  i> 


INTRODUCTION.  15 

of  particular  importance,  not  only  because  of  the  careful, 
objective  nature  of  the  description  given  for  the  bodies 
seen  by  him,  but  also  for  the  illustrations  which  accom- 
pany it.  From  a  perusal  of  the  text  and  an  inspection 
of  the  plates  there  remains  little  room  for  doubt  that 
Leeuwenhoek  with  his  primitive  lens  had  seen  the  bodies 
now  recognized  as  bacteria. 

Upon  seeing  these  bodies  he  was  apparently  very 
much  astonished,  for  he  writes:  "With  the  greatest 
astonishment  I  saw  that  everywhere  through  the  ma- 
terial which  I  was  examining  were  distributed  animal- 
cules of  the  most  microscopic  dimensions,  which  moved 
themselves  about  in  a  remarkably  energetic  way." 

This  observation  was  shortly  followed  by  others  of 
an  equally  important  nature.  His  field  of  observation 
appears  to  have  increased  rapidly,  for  after  a  time  he 
speaks  of  bodies  of  much  smaller  dimensions  than  those 
at  first  described  by  him. 

Throughout  all  of  Leeuwenhoek's  work  there  is  a 
conspicuous  absence  of  the  speculative.  His  contribu- 
tions are  marked  by  their  purely  objective  nature. 

After  the  presence  of  these  organisms  in  water,  in  the 
mouth,  and  in  the  intestinal  evacuations  was  made 
known  to  the  world,  it  is  hardly  surprising  that  they 
were  immediately  seized  upon  as  the  explanation  for  the 
origin  of  many  obscure  diseases.  So  universal  was  the 
belief  in  a  causal  relation  between  these  "  animalcules  " 
and  disease,  that  it  amounted  almost  to  a  germ  mania. 
It  became  the  fashion  to  suspect  the  presence  of  these 
organisms  in  all  forms  and  kinds  of  disease,  simply 
because  they  had  been  demonstrated  in  water. 

Though  nothing  of  value  at  the  time  had  been  done 
in  the  way  of  classification,  and  still  less  in  separating 


16  BACTERIOLOGY. 

and  identifying  the  members  of  this  large  group,  still, 
the  foremost  men  of  the  day  did  not  hesitate  to  ascribe 
to  them  not  only  the  property  of  producing  disease  con- 
ditions, but  some  even  went  so  far  as  to  hold  that  varia- 
tions in  the  appearance  of  the  symptoms  of  disease  were 
the  result  of  differences  in  the  behavior  of  the  organisms 
in  the  tissues. 

Marcus  Antonius  Plenciz,  a  physician  of  Vienna  in 
1762,  expressed  himself  a  firm  believer  in  the  work  of 
Leeuwenhoek,  and  based  the  doctrine  which  he  taught 
upon  the  discoveries  of  the  Dutch  observer,  and  upon 
observations  of  a  confirmatory  nature  which  he  him- 
self had  made.  The  doctrine  of  Plenciz  assumed  a 
causal  relation  between  the  microorganisms  discovered 
and  described  by  Leeuwenhoek  and  all  infectious  dis- 
eases. He  claimed  that  infection  could  be  nothing  else 
than  a  living  substance,  and  endeavored  on  these  grounds 
to  explain  the  variations  in  the  period  of  incubation  for 
the  different  infectious  diseases.  He  likewise  believed 
the  living  contagium.  to  be  capable  of  multiplication 
within  the  body,  and  spoke  of  the  possibility  of  its 
transmission  through  the  air.  He  claimed  a  special 
germ  for  each  disease,  holding  that  just  as  from  a  given 
cereal  only  one  kind  of  grain  can  grow,  so  by  the  special 
germ  for  each  disease  ouly  that  disease  can  be  produced. 

He  found  in  all  decomposing  matters  innumerable 
minute  "  animalculse,"  and  was  so  firmly  convinced  of 
their  etiological  relation  to  the  process  that  he  formu- 
lated the  law  :  that  decomposition  can  only  take  place 
when  the  decomposable  material  becomes  coated  with  a 
layer  of  the  organisms,  and  can  proceed  only  when  they 
increase  and  multiply. 

However  convincing  the  arguments  of  Plenciz  appear, 


INTRODUCTION.  17 

they  seem  to  have  been  lost  sight  of  in  the  course  of 
subsequent  events,  and  by  a  few  were  even  held  in  the 
light  of  productions  from  an  unbalanced  mind.  For 
example,  as  late  as  1820  we  find  Ozanam  expressing 
himself  on  the  subject  as  follows  :  "  Many  authors  have 
written  concerning  the  animal  nature  of  the  contagion 
of  infectious  diseases ;  many  have  indeed  assumed  it  to 
be  developed  from  animal  substances  and  that  it  is  itself 
animal,  and  possesses  the  property  of  life.  I  shall  not 
waste  time  in  efforts  to  refute  these  absurb  hypotheses." 

Similar  expressions  of  opinion  were  heard  from  many 
other  medical  men  of  the  time,  all  tending  in  the  same 
direction,  all  doubting  the  possibility  of  these  micro- 
scopic creatures  belonging  to  the  world  of  living  things. 

It  was  not  until  between  the  fourth  and  fifth  decade 
of  the  present  century  that  by  the  fortunate  coincidence 
of  a  number  of  important  discoveries  the  true  relation 
of  the  lower  organisms  to  infectious  diseases  was  scien- 
tifically pointed  out.  With  the  investigations  of  Pasteur 
upon  the  cause  of  putrefaction  in  beer  and  the  souring 
of  wine ;  with  the  discovery  by  Pollender  and  Davaine 
of  the  presence  of  rod-shaped  organisms  in  the  blood  of 
all  animals  dead  of  splenic  fever,  and  the  progress  of 
knowledge  upon  the  parasitic  nature  of  certain  diseases 
of  plants,  the  old  question  of  "  contagium  animatum" 
again  began  to  receive  attention.  It  was  taken  up  by 
Henle,  and  it  was  he  who  first  logically  taught  this  doc- 
trine of  infection. 

The  main  point,  however,  which  had  occupied  the 
attention  of  scientific  men  from  time  to  time  for  a 
period  of  about  two  hundred  years  subsequent  to 
Leeu  wen  hock's  discoveries,  was  the  origin  of  these 
bodies.  Do  they  generate  spontaneously,  or  are  they 


18  BACTERIOLOGY. 

the  descendants  of  preexisting  creatures  of  the  wime 
kind?  was  the  all-important  question.  Among  the 
participants  in  this  discussion  were  many  of  the  most 
prominent  men  of  the  day. 

In  1749  Needham,  who  held  firmly  to  the  opinion 
that  the  bodies  which  were  creating  such  a  general 
interest  developed  spontaneously,  as  the  result  of  vege- 
tative changes  in  the  substances  in  which  they  were 
found,  attempted  to  demonstrate  by  experiment  tin- 
grounds  upon  which  he  held  this  view.  He  maintained 
that  the  bacteria  which  were  seen  to  appear  around  a 
grain  of  barley  which  was  allowed  to  germinate  in  a 
watch-crystal  of  water,  which  had  been  carefully  cov- 
ered, were  the  result  of  changes  in  the  barley-grain 
itself,  incidental  to  its  germination. 

Spallauzani,  in  1769,  drew  attention  to  the  laxity  of 
the  methods  employed  by  Needham,  and  demonstrated 
that  if  infusions  of  decomposable  vegetable  matter  were 
placed  in  flasks,  which  were  then  hermetically  sealed, 
and  the  flasks  and  their  contents  allowed  to  remain  for 
some  time  in  a  vessel  of  boiling  water,  neither  living 
organisms  could  be  detected  nor  would  decomposition 
appear  in  the  infusions  so  treated.  The  objection  raised 
by  Treviranus  that  the  high  temperature  to  which  the 
infusions  had  been  subjected  had  so  altered  them  and  the 
air  about  them,  that  the  conditions  favorable  to  spon- 
taneous generation  no  longer  existed,  was  met  by  Spal- 
lanzaui  by  gently  tapping  one  of  the  flasks  that  had  been 
boiled,  against  some  hard  object  until  a  minute  crack 
was  produced  ;  invariably  organisms  and  decomposition 
appeared  in  the  flask  thus  treated. 

From  the  time  of  the  experiments  of  Spallanzani 
until  as  late  as  1836  but  little  advance  was  made  in  the 
elucidation  of  this  obscure  problem. 


IXTliODUCTION.  19 

In  1836  Sehulz  attracted  attention  to  the  subject  by 
the  convincing  nature  of  his  investigations.  He  showed 
that  if  the  air  which  gained  access  to  boiled  infusions 
was  robbed  of  its  living  organisms  by  being  caused  to 
pass  through  strong  acid  or  alkaline  solutions,  no  de- 
composition appeared  and  living  organisms  could  not 
be  detected  in  the  infusions.  Following  quickly  upon 
this  contribution  came  Schwann,  in  1837,  and  somewhat 
later  (1854)  Schroder  and  Dusch,  with  similar  results  ob- 
tained by  somewhat  different  means.  Schwann  deprived 
the  air  which  passed  to  his  infusions  of  its  living  particles 
by  passing  it  through  highly-heated  tubes;  whereas 
Schroder  and  Dusch,  by  means  of  cotton- wool  interposed 
between  the  boiled  infusion  and  the  outside  air,  robbed 
the  air  passing  to  the  infusions  of  its  organisms  by  the 
simple  process  of  filtration.  In  1860  Hoffmann  and  in 
1861  Chevreul  and  Pasteur  demonstrated  that  the  pre- 
cautions taken  by  the  preceding  investigators  for  ren- 
dering the  air  which  entered  these  flasks  free  from 
bacteria  were  not  necessary  ;  that  all  that  was  necessary 
to  prevent  the  access  of  bacteria  to  the  infusions  in  the 
flasks  was  to  draw  out  the  neck  of  the  flask  into  a  fine 
tube,  bend  it  down  along  the  side  of  the  flask  and  then 
bend  it  up  again  a  few  inches  from  its  extremity,  and 
leave  the  mouth  open.  The  infusion  was  then  to  be 
boiled  in  the  flask  thus  prepared  and  the  mouth  of  the 
tube  left  open.  The  organisms  which  now  fall  into  the 
tube  will  be  arrested  by  the  drop  of  water  of  condensa- 
tion which  collects  at  its  lowest  angle,  and  none  can 
enter  the  flask. 

Convincing  though  this  work  may  seem,  there  still 
existed  a  number  of  doubters  who  required  further  proof 
that  "  spontaneous  generation  "  was  not  the  explanation 


20  BACTERIOLOGY. 

for  the  mysterious  appearance  of  these  minute  living 
objects,  and  it  was  not  until  some  time  later  that  Tyn- 
dall,  in  his  well-known  investigations  upon  the  floating 
matters  in  the  air,  demonstrated  again  that  the  presence 
of  living  organisms  in  decomposing  fluids  was  always 
to  be  explained  either  by  the  preexistence  of  similar  liv- 
ing forms  in  the  infusion  or  upon  the  walls  of  the  vessel 
containing  it,  or  by  the  infusion  having  been  exposed 
to  air  which  had  not  been  deprived  of  its  orgauisms. 

Throughout  all  the  work  bearing  upon  this  subject, 
from  the  time  of  Spallanzani  to  that  of  Tyndall,  certain 
irregularities  were  constantly  appearing.  It  was  found 
that  certain  substances  required  to  be  heated  for  a  much 
longer  time  than  was  necessary  to  render  other  sub- 
stances free  from  living  organisms,  and  even  under  the 
most  careful  precautions  decomposition  would  occasion- 
ally appear. 

In  1762  Bonnet,  who  was  deeply  interested  in  this 
subject,  had  suggested,  in  reference  to  the  results  ob- 
tained by  Needham,  the  possibility  of  the  existence  of 
"  germs,  or  their  eggs,"  which  have  the  power  to  resist 
the  temperature  to  which  some  of  the  infusions  employed 
in  Needham's  experiments  had  been  subjected. 

More  than  a  hundred  years  after  Bonnet  had  made 
this  purely  speculative  suggestion,  it  became  the  task  of 
Ferdinand  Cohn,  of  Breslau,  to  demonstrate  its  accuracy. 

Cohn  repeated  the  foregoing  experiments  with  like 
results.  He  concluded  that  the  irregularities  could  only 
i)o  due  to,  either  the  existence  of  more  resistant  species 
of  bacteria  or  to  more  resistant  stages  into  which  certain 
bacteria  have  the  property  of  passing.  After  much 
work  he  demonstrated  that  certain  of  the  rod-shaped 
organisms  possess  the  power  of  passing  into  a  resting 


INTRODUCTION.  21 

or  spore  stage  in  the  course  of  their  life  history,  and 
when  in  this  stage  they  are  much  less  susceptible  to  the 
deleterious  action  of  high  temperatures  than  when  they 
are  growing  as  normal  vegetative  forms.  With  the 
discovery  of  these  more  resistant  spores,  the  doctrine  of 
spontaneous  generation  received  its  final  blow.  It  was  no 
longer  difficult  to  explain  the  irregularities  in  the  fore- 
going experiments,  or  was  it  any  longer  to  be  doubted  that 
putrefaction  and  fermentation  were  the  result  of  bacterial 
life  and  not  the  cause  of  it,  and  that  these  bacteria  were 
the  offspring  from  preexisting  similar  forms.  In  other 
words,  the  "law  of  Harvey,  Omne  vivum  ex  ovo,  or  its 
modification,  Omne  vivum  ex  vivo,  was  shown  to  apply, 
not  only  to  the  more  highly  organized  members  of  the 
animal  and  vegetable  kingdoms,  but  to  the  most  micro- 
scopic, unicellular  creatures  as  well. 

NOTE. — I  have  presented  only  the  most  prominent 
investigations  which  aided  in  overthrowing  the  doctrine 
of  spontaneous  generation.  For  a  more  detailed  account 
of  this  work  the  reader  is  referred  to  Loffler's  Vorle- 
sungen  uber  die  geschichtliche  Entwickelung  der  Lehre 
von  den  Bacterien,  upon  which  I  have  drawn  largely  in 
preparing  the  foregoing  sketch. 


2* 


CHAPTER    I. 

Definition  of  bacteria— Their  place  in  nature— Difference  between 
parasites  and  saprophytes — Nutrition  of  bacteria — Product-  of  bac- 
teria— Their  relation  to  oxygen — Influence  of  temperature  upon 
their  growth. 

BY  the  term  bacteria  is  understood  that  large  group 
of  minute  vegetable  organisms  which  multiply  by  a  pro- 
cess of  transverse  division.  They  are  spherical,  oval, 
rod-like,  and  spiral  in  shape,  and  are  commonly  devoid 
of  chlorophyll.1  Owing  to  the  absence  of  chlorophyll 
from  their  composition  the  bacteria  are  forced  to  either  a 
saprophytic2  or  parasitic3  form  of  existence. 

Their  life  processes  are  so  rapid  and  energetic  that 
they  result  in  the  most  profound  alterations  in  the 
structure  and  composition  of  the  materials  upon  which 
they  are  developing. 

Decomposition  and  fermentation  result  from  the  pres- 
ence of  the  saprophytic  bacteria,  while  the  changes 

1  Chlorophyll  is  the  green  coloring  matter  possessed  by  the  higher 
plants  by  means  of  which  they  are  enabled  in  the  presence  of  sun- 
light to  decompose  carbonic  acid  (CO2X)  and  ammonia   (NIT.,)  into 
their  elementary  constituents. 

2  A  saprophyte  is  an  organism    that  obtains  its  nutrition    from 
dead  organic  matter. 

3  A  parasite  lives  always  at  the  expense  of  some  other  living, 
organic  creature,  and  in  the  strictest  sense  of  the  word  cannot  exist 
upon  dead  matter.     There  exist,   however,  a  group   of  so-called 
"facultative"  saprophytes  and  para-it<--  which  possess  the  power  of 
accommodating  themselves  to   existing  surroundings — at  one  time 
leading  a  parasitic,  at  another  time  a  saprophytic  form  of  existence. 


PARASITES    AND    SAPROPHYTES.  23 

brought  about  in  the  tissues  of  their  host  by  the  pure 
parasitic  forms,  find  expression  in  disease  processes  and 
not  unfrequently  complete  death. 

The  role  played  in  nature  by  the  saprophytic  bacteria 
is  a  very  important  one.  Through  their  presence  the 
highly  complicated  tissues  of  dead  animals  and  vegeta- 
bles are  resolved  into  the  simpler  compounds,  carbonic 
acid  and  ammonia,  in  which  form  they  may  be  taken  up 
and  appropriated  as  nutrition  by  the  more  highly  organ- 
ized members  of  the  vegetable  kingdom.  It  is  to  this 
ultimate  production  of  carbonic  acid,  ammonia,  and  water 
by  the  bacteria,  as  end-products  in  the  processes  of  de- 
composition and  fermentation  of  the  dead  animal  and 
vegetable  tissues,  that  the  demands  of  growing  vegeta- 
tion for  these  compounds  can  be  supplied. 

The  chlorophyll  plants  do  not  possess  the  power  of 
obtaining  their  carbon  and  nitrogen  from  such  highly 
organized  and  complicated  substances  as  serve  for  the 
nutrition  of  the  bacteria,  and  as  the  production  of  these 
simpler  compounds  (CO2,  NH3,  H20)  by  the  animal 
world  is  not  sufficient  to  meet  the  demands  of  the  chloro- 
phyll plants,  the  importance  of  the  part  played  by  the 
bacteria  in  making  up  this  deficit  cannot  be  overestimated. 
Were  it  not  for  the  activity  of  these  microscopic  living 
particles,  all  life  upon  the  surface  of  the  earth  would 
certainly  cease.  Deprive  higher  vegetation  of  the  car- 
bon and  nitrogen  supplied  to  it  as  a  result  of  bacterial 
activity,  and  its  development  comes  rapidly  to  an  end. 
Rob  the  animal  kingdom  of  the  food-stuffs  supplied  to 
it  by  the  vegetable  world,  and  life  is  no  longer  possible. 

It  is  plain,  therefore,  that  the  saprophytes,  which  re- 
present by  far  the  large  majority  of  all  bacteria,  must  be 


24  BACTERIOLOGY. 

looked  upon  by  us  in  the  light  of  benefactors,  without 
which  existence  would  be  impossible. 

With  the  parasites,  on  the  other  hand,  the  condition- 
are  far  from  analogous.  Through  their  existence  there 
is  constantly  a  loss,  rather  than  a  gain,  to  both  the 
animal  and  vegetable  kingdoms.  Their  host  must 
always  be  a  living  body  in  which  exist  conditions  favor- 
able to  their  development  and  from  which  they  appro- 
priate substances  which  are  necessary  to  the  health  and 
life  of  the  tissues  of  the  organism  to  which  they  may 
have  found  access.  At  the  same  time  the  substances 
which  they  form  as  products  of  their  nutrition  are  direct 
poisons  for  the  surrounding  tissues. 

In  their  relations  to  humanity  the  positions  occupied 
by  the  two  biologically  different  groups,  the  saprophytes 
on  the  one  hand  and  the  parasites  on  the  other,  are 
diametrically  opposite.  The  saprophytic  forms  standing 
in  the  relation  of  benefactors,  in  resolving  dead  animal 
and  vegetable  bodies  into  their  component  parts,  which 
serve  for  food  for  living  vegetation,  and,  at  the  same 
time,  removing  from  the  surface  of  the  earth  the  re- 
mains of  all  dead  organic  substances  ;  while  the  parasitic 
group  exist  only  at  the  expense  of  the  more  highly 
organized  members  of  both  kingdoms.  It  is  to  the 
parasitic  group  that  the  pathogenic1  organisms  belong. 
*  In  addition  to  the  saprophytes  which  are  concerned 
in  the  changes  to  which  allusion  has  just  been  made, 
there  exist  other  saprophytic  forms  which  are  recognized 
by  their  property  of  producing  pigments  of  different 
color.  These  are  known  as  the  chromogenic2  forms. 

1  Pathogenic  organisms  are  those  which  possess  the  property  of 
producing  disease. 
s  Chromogenic,  possessing  the  property  of  producing  color. 


NUTRITION    OF    BACTERIA.  25 

Just  what  their  exact  role  in  nature  is,  it  is  difficult  to 
say  ;  but  it  is  probable  that  in  addition  to  their  most 
conspicuous  function  of  color  production,  they  are  also  in 
some  way  concerned  in  the  great  process  of  disintegra- 
tion which  is  constantly  going  on  in  all  dead  organic 
substances. 

We  know  that  through  the  agency  of  chlorophyll,  in 
the  presence  of  sunlight,  the  green  plants  are  enabled  to 
obtain  the  amount  of  nitrogen  and  carbon  which  is  neces- 
sary to  their  growth  from  such  simple  bodies  as  carbon 
dioxide  and  ammonia,  which  they  decompose  into  their 
elementary  constituents.  The  bacteria,  on  the  other 
hand,  owing  to  the  absence  of  chlorophyll  from  their 
tissues,  do  not  possess  this  power.  They  must  have 
their  carbon  and  nitrogen  presented  as  such,  in  the 
form  of  decomposable  organic  compounds. 

In  general,  the  bacteria  obtain  their  nitrogen  most 
readily  from  soluble  albumins,  and,  to  a  certain  degree, 
but  by  no  means  so  easily,  from  salts  of  ammonia.  In 
some  of  Nageli's  experiments  it  appeared  probable  that 
they  could  obtain  the  necessary  amount  of  nitrogen  from 
the  salts  of  nitric  acid.  At  all  events,  he  was  able  in 
certain  cases  to  demonstrate  a  reduction  of  nitric 
to  nitrous  acid,  and  ultimately  to  ammonia.  Neverthe- 
less, in  all  of  these  experiments  circumstances  point  to 
the  probability  that  the  nitrogen  obtained  by  the  bacteria 
for  building  up  their  tissues  in  the  course  of  their  devel- 
opment, was  derived  from  some  source  other  than  that  of 
the  nitric  acid  or  the  nitrites,  and  that  the  reduction  of 
this  acid  was  most  probably  a  secondary  phenomenon. 

For  the  supply  of  carbon,  many  of  the  carbon  com- 
pounds serve  as  sources  upon  which  the  bacteria  can 
draw.  The  carbon  deficit,  for  example,  can  be  obtained 


26  BACTERIOLOGY. 

from  sugar  and  bodies  of  like  composition  ;  from  glyce- 
rine and  many  of  the  fatty  acids ;  and  from  the  alkaline 
salts  of  tartaric,  citric,  malic,  lactic,  and  acetic  acids.  In 
some  instances  carbon  compounds  which  when  present 
in  concentrated  form  inhibit  the  growth  of  the  lower 
organisms,  may,  when  highly  diluted,  serve  as  nutrition 
for  these  bodies.  Salicylic  acid  and  ethyl  alcohol  come 
under  this  head. 

In  addition  to  carbon  and  nitrogen,  water  is  essential 
to  the  life  and  development  of  bacteria.  Without  it 
no  development  occurs,  and  in  many  cases  drying  the 
organisms  results  in  their  death.  Certain  forms,  on  the 
contrary,  though  incapable  of  multiplying  when  in  the 
dry  stage,  may  be  completely  deprived  of  their  water 
without  causing  them  to  lose  the  power  of  reproduction 
when  favorable  conditions  present. 

The  closer  study  of  the  bacteria,  and  a  more  intimate 
acquaintance  with  their  nutritive  changes,  demonstrate 
an  appreciable  variability  in  the  character  of  the  sub- 
stances best  suited  for  the  nutrition  of  different  species, 
one  requiring  a  more  concentrated  form  of  nutrition, 
while  another  needs  but  a  very  limited  amount  of  pro- 
teid  substance  for  its  development.  Certain  members 
bring  about  most  profound  alterations  in  the  media  in 
which  they  exist,  while  others  produce  but  little  appa- 
rent change.  In  one  case  alterations  in  the  reaction  of 
the  media  will  be  most  conspicuous,  while  in  another  no 
such  variation  can  be  detected.  With  certain  forms 
oxygen  is  essential  for  the  proper  performance  of  their 
functions,  while  with  another  group  no  evidence  of  life 
can  be  detected  under  the  access  of  oxygen,  and  in  a 
third  group  oxygen  appears  to  play  but  an  unimportant 
part,  for  development  occurs  as  well  with  as  without 


BACTERIAL    RELATION    TO    OXYGEN.          27 

it.  In  the  case  of  certain  of  the  chromogenic  forms 
the  presence  or  absence  of  oxygen  has  a  very  decided 
effect  upon  the  production  of  the  pigments  by  which 
they  are  characterized. 

For  the  normal  development  of  bacteria  it  is  not  only 
essential  that  the  sources  from  which  they  can  obtain  the 
necessary  nutritive  elements  should  exist,  but  account 
must  also  be  taken  of  the  products  of  growth  of  the 
organism  in  these  substances.  Nitrogen  and  carbon 
compounds  in  the  proper  form  to  be  taken  up  and  appro- 
priated by  the  organism  may  exist  in  sufficient  quanti- 
ties, and  still  the  growth  of  the  organism  after  a  very 
short  time  be  entirely  checked,  owing  to  the  production 
during  their  growth  of  substances  inhibitory  to  their  fur- 
ther development.  Most  conspicuous  are  the  changes 
produced  by  the  growing  bacteria  in  the  reaction  of  the 
media.  Since  the  majority  of  these  bodies  grow  best  in 
media  of  a  neutral  or  very  slightly  alkaline  reaction,  any 
excessive  production  of  alkalinity  or  acidity,  as  a  pro- 
duct of  growth,  arrests  development,  and  no  evidence  of 
life  or  further  multiplication  can  be  detected  until  this 
deviation  from  the  neutral  reaction  has  been  corrected. 

Most  favorable  for  the  development  of  bacteria  are 
neutral  or  very  slightly  alkaline  solutions  of  albumin  in 
one  form  or  another. 

Of  considerable  importance  and  interest  in  the  study 
of  the  nutritive  changes  of  bacteria  is  the  difference  in 
their  relation  to  oxygen.  It  was  Pasteur  who  first 
demonstrated  the  existence  of  species  in  the  bacteria 
family  which  not  only  grow  and  multiply  and  per- 
form definite  physiological  functions  without  the  aid 
of  oxygen,  but  to  the  existence  of  which  oxygen  is 
positively  harmful.  To  these  he  gave  the  name  of 


28  BACTERIOLOGY. 

"anaerobic"  bacteria  in  contradistinction  to  another 
group  for  the  proper  performance  of  whose  functions 
"\\<ren  is  essential,  which  he  called  "aerobic"  bacteria. 
In  addition  to  these,  there  is  a  third  group  for  the  main- 
tenance of  whose  existence  the  absence  or  presence  of 
oxygen  is  apparently  of  no  moment — their  development 
progresses  as  well  with  as  without  it ;  these  represent 
the  class  known  as  "facultative"  in  their  relation  to 
this  gas.  It  is  in  this  third  group,  the  facultative,  that 
the  majority  of  bacteria  belong.  Though  the  multipli- 
cation of  the  facultative  varieties  is  not  interfered  with 
by  either  the  presence  or  absence  of  oxygen,  yet  experi- 
ments show  that  the  products  of  their  growth  are  differ- 
ent under  the  varying  conditions  of  absence  or  presence 
of  this  gas. 

Another  element  which  plays  a  most  important  part 
in  the  biological  functions  of  these  organisms  is  the 
temperature  under  which  they  exist.  The  extremes  of 
temperature  under  which  bacteria  are  known  to  grow 
range  from  5.5°  C.  to  48°  C.  At  the  former  tempera- 
ture development  is  hardly  appreciable,  it  becomes  more 
and  more  active  until  38°  C.  is  reached,  when  it  is  at  its 
optimum,  and,  as  a  rule,  ceases  with  43°  C.  Neither  of 
the  extremes  can  be  considered  normal  temperatures  for 
the  growth  of  these  organisms.  The  most  favorable 
temperature  for  the  development  of  the  majority  of 
bacteria  is  that  of  the  human  body,  viz.,  37.5°  C. 

In  general  it  may  be  said  that  for  the  growth  and 
development  of  bacteria,  organic  matter  of  a  neutral  or 
slightly  alkaline  reaction,  in  the  presence  of  moisture 
and  at  a  suitable  temperature,  is  necessary.  From  this 
can  be  formed  some  idea  of  the  omnipresence  in  nature 
of  these  minute  vegetable  forms.  Everywhere  that 
these  conditions  exist,  bacteria  can  be  found. 


CHAPTER    II. 

Morphology1  of  the  bacteria — Grouping — Mode  of  multiplication 
— Spore-formation — Motility. 

IN  form  the  bacteria  are  unicellular,  and  are  seen  to 
exist  as  spherical,  rod-  or  spindle-shaped  bodies.  They 
always  develop  from  preexisting  cells  of  the  same  char- 
acter and  never  appear  spontaneously. 

The  classifications  of  the  older  authors  were  upon 
purely  morphological  peculiarities,  and  in  consequence, 
were  more  or  less  complicated.  The  present  tendency 
is  to  simplify  this  morphological  classification,  and  to 
bring  the  bacteria  into  three  great  groups,  with  their 
subdivisions ;  each  group  comprising  those  members 
whose  individual  outline  is  that  either  of  a  sphere,  a 
rod,  or  a  spiral. 

To  these  three  grand  divisions  are  given  the  names 
cocci  or  micrococci,  bacilli,  and  spirilli. 

In  the  group  micrococci  belong  all  spherical  forms, 
i.  e.,  all  those  forms  the  individual  members  of  which 
are  of  equal  diameter  in  all  directions. 

The  bacilli  comprise  all  oval  or  rod-formed  bacteria. 

To  the  spirilli  belong  all  organisms  which  are  twisted 
in  the  form  of  a  corkscrew. 

The  micrococci  are  subdivided  according  to  their 
grouping,  as  seen  in  growing  cultures  :  into  staphylo- 
cocci — those  growing  in  masses  like  fish-roe  or  clusters 
of  grapes ;  streptococci — those  growing  in  chains  con- 

1  Morphology,  pertaining  to  shape ;  outline. 


30  BACTERIOLOGY. 

sisting  of  a  number  of  individual  cells  strung  together 
like  beads  or  pearls  upon  a  string ;  diplococci — those 
growing  in  pairs ;  tetrads — those  developing  as  fours, 
and  sarcince — those  dividing  into  fours,  eights,  etc.,  as 
cubes — that  is,  in  contra-distinction  to  all  other  forms, 
the  segmentation,  which  is  rarely  complete,  takes  place 
in  three  directions  of  space,  so  that  when  growing,  the 
bundle  of  segmenting  cells  presents  somewhat  the  ap- 
pearance of  a  bale  of  cotton. 

To  the  bacilli  belong  all  rod-shaped  organisms,  /.  e., 
those  in  which  one  diameter  is  always  greater  than  the 
other. 

In  this  group  are  found  those  organisms  the  life  cycle 
of  many  of  which  present  deviations  from  the  simple 
rod  shape.  Many  of  them  in  the  course  of  development 
increase  in  length  into  long  threads  along  the  course  of 
which  traces  of  segmentation  may  usually  be  found — 
the  anthrax  bacillus  and  bacillus  subtilis  are  conspicuous 
examples  of  this.  Again  others,  under  certain  condi- 
tions, possess  the  property  of  forming  within  the  body 
of  the  rods  oval,  glistening  spores,  and  if  the  conditions 
are  not  altered  the  rods  may  entirely  disappear,  so  that 
nothing  may  be  left  in  the  culture  but  these  oval  forms. 
Again,  many  of  them,  from  unfavorable  conditions  of 
nutrition,  aeration,  or  temperature  undergo  pathological 
changes — that  is,  the  individuals  themselves  experience 
alterations  in  their  protoplasm  which  result  in  distor- 
tion of  their  outline.  This  is  the  production  of  the 
so-called  "  involution  forms."  But  in  all  of  these  con- 
ditions, so  long  as  death  has  not  actually  occurred,  it  is 
possible  under  favorable  conditions  to  cause  these  forms 
to  revert  to  the  original  rod-shaped  bacilli  from  which 
they  originated. 


MORPHOLOGY.  81 

It  must  be  borne  in  mind,  however,  that  it  is  never 
possible  by  any  means  to  bring  about  changes  in  these 
organisms  that  will  result  in  the  permanent  conversion 
of  the  morphology  of  the  members  of  one  group  into 
that  of  another — that  is,  one  can  never  produce  bacilli 
from  micrococci  or  vice  versa,  and  any  evidence  which 
may  be  presented  to  the  contrary  is  based  upon  inaccu- 
rate methods  of  work. 

Not  infrequently  bacilli  may  be  observed  irregularly 
massed  together  as  a  pellicle.  When  in  this  condition 
they  are  held  together  by  a  gelatinous  material,  and  are 
known  as  zoogloea  of  bacilli. 

Very  short  oval  bacilli  may  sometimes  be  mistaken  for 
micrococci,  and  at  times  micrococci  in  the  stage  of  segmen- 
tation into  diplococci  may  be  mistaken  for  short  bacilli ; 
but  by  careful  inspection  it  will  always  be  possible  to  de- 
tect a  continuous  outline  along  the  sides  of  the  former 
and  a  slight  transverse  indentation  or  partition-forma- 
tion between  the  segments  of  the  latter.  The  high  index 
of  refraction  of  spores,  the  property  which  gives  to 
them  their  glistening  appearance,  will  always  serve  to 
distinguish  them  from  micrococci.  This  difference  in 
refraction  will  be  especially  noticed  if  the  illumination 
from  the  reflector  of  the  microscope  writh  which  they  are 
to  be  examined  is  reduced  to  the  smallest  possible  bundle 
of  light-rays.  The  spores,  moreover,  take  up  the  staining 
reagents  much  less  readily  than  do  the  micrococci.  The 
crucial  test,  however,  is  the  property,  in  the  case  of  the 
spores,  of  growing  out  into  bacilli;  and  of  the  spherical 
organism  with  which  it  has  been  confounded,  of  develop- 
ing only  into  another  micrococcus  of  the  same  round 
form. 

For  convenience,  a  common  classification  of  the  bacilli 


32  BACTERIOLOGY. 

is  that  based  upon  constant  characteristics  which  are 
seen  to  appear  in  the  course  of  their  development  under 
special  conditions — certain  of  them  possessing  the  power 
of  forming  spores,  while  from  others  this  peculiarity  is 
absent. 

As  yet  but  little  is  known  of  the  life  history  of  the 
spiral  forms.  Efforts  toward  their  cultivation  under 
artificial  conditions  have  thus  far  been  unsuccessful. 
Morphologically,  they  are  thread-  or  rod-like  bodies 
which  are  tsvisted  into  the  form  of  spirals.  In  some  of 
them  the  turns  of  the  spiral  are  long,  in  others  quite 
short.  They  are  motile,  and  multiply  apparently  by 
the  simple  process  of  fission.1 

The  micrococci  develop  by  simple  fission.  When 
development  is  in  progress  a  single  cell  will  be  seen  to 
elongate  slightly  in  one  of  its  diameters.  Over  the 
center  of  the  long  axis  thus  formed  will  appear  a  slight 
indentation  in  the  outer  envelope  of  the  cell ;  this 
indentation  will  increase  in  extent  until  there  exist 
eventually  two  individuals  which  are  distinctly  spheri- 
eal,  as  was  the  parent  from  which  they  sprang,  or  they 
will  remain  together  for  a  time  as  diplococci.  The  sur- 
faces now  in  juxtaposition  are  flattened  against  one 
another,  and  not  infrequently  a  fine,  pale  dividing  line 
may  be  seen  between  the  two  cells.  A  similar  division 
in  the  other  direction  will  now  result  in  the  formation 
of  a  group  of  forms  as  tetrads.  This,  in  short,  is  the 
method  of  multiplication  of  the  micrococci. 

In  the  formation  of  the  staphylococci  such  division 
occurs  irregularly  in  all  directions,  resulting  in  the  pro- 
duction of  the  clusters  in  which  these  organs  are  coni- 

1    Dividing  into  i\\«>  tr:iiiMvr>«-lv. 


MODE    OF    MULTIPLICATION.  33 

mouly  seen.  With  the  streptococci,  however,  the  tendency 
is  for  the  segmentation  to  continue  in  one  direction  only, 
resulting  in  the  production  of  the  long  chains  of  4,  8, 
and  12  individuals. 

The  sarcinse  divide  more  or  less  regularly  in  three 
directions  of  space,  but  instead  of  becoming  separated 
the  one  from  the  other  as  single  cells,  the  tendency  is 
for  the  segmentation  to  be  incomplete.  The  cells  remain 
together  in  masses  and  the  indentations  upon  these  masses 
or  cubes  which  indicate  the  point  of  incomplete  fission 
give  to  these  bundles  of  cells  the  appearance  commonly 
ascribed  to  them — that  of  a  bale  of  cotton  or  packet  of 
rags. 

The  multiplication  of  the  bacilli  is  in  the  main  similar 
to  that  given  for  the  micrococci.  A  dividing  cell  will 
elongate  slightly  in  the  direction  of  its  long  axis;  an 
indentation  will  appear  about  midway  between  its  poles, 
and  will  become  deeper  and  deeper  until  eventually  two 
daughter  cells  will  be  formed.  This  process  may  occur 
in  such  a  way  that  the  two  young  bacilli  will  adhere 
together  by  their  adjacent  ends  in  much  the  same  way 
that  sausages  are  seen  to  be  held  together  in  strings,  or 
the  segmentation  may  take  place  more  at  right  angles  to 
the  long  axis,  so  that  the  proximal  ends  of  the  young 
cells  are  flattened  while  the  distal  extremities  may  be 
rounded  or  slightly  pointed.  In  the  anthrax  bacillus, 
with  which  we  are  subsequently  to  become  more  inti- 
mately acquainted,  the  segmentation,  when  completed, 
results  in  an  indentation  of  the  adjacent  extremities  of 
the  young  segments,  so  that  by  the  aid  of  high  magni- 
fying powers  these  surfaces  are  seen  to  be  actually  con- 
cave in  their  outline.  Bacilli  never  divide  longitudinally. 

With  the  spore-forming  bacilli,  under  favorable  con- 


34  BACTERIOLOGY. 

ditions  of  nutrition  and  temperature,,  the  same  process  is 
seen  to  occur,  but  so  soon  as  these  conditions  become 
altered,  either  by  the  exhaustion  of  the  nutrition,  the 
presence  of  detrimental  substances,  unfavorable  tempera- 
tures, etc.,  there  appears  the  stage  in  their  life  cycle  to 
which  we  have  referred  as  "spore-formation."  This  is 
the  process  by  which  the  organisms  are  enabled  to  enter 
a  stage  in  which  they  resist  deleterious  influences  to  a 
much  higher  degree  than  is  possible  for  them  when  in 
the  growing  or  vegetative  condition. 

In  the  spore,  resting,  or  permanent  stage,  as  it  is 
called,  no  evidence  of  life  whatever  is  given  by  the 
spores,  though  as  soon  as  the  conditions  which  favor 
their  germination  have  been  renewed,  these  spores  de- 
velop again  into  the  same  kind  of  cells  as  those  from 
which  they  originated,  and  the  appearances  observed  in 
the  vegetative  or  growing  stage  of  their  history  are  again 
to  be  seen. 

Multiplication  of  spores,  as  such,  .does  not  occur. 
They  possess  the  power  of  developing  into  individual 
rods  of  the  same  nature  as  those  from  which  they  were 
formed,  but  not  of  giving  rise  to  a  direct  reproduction 
of  spores. 

When  the  conditions  which  favor  spore-formation 
present,  the  protoplasm  of  the  vegetative  cells  is  seen 
to  undergo  a  change.  It  loses  its  normal  homogeneous 
appearance  and  becomes  marked  here  and  there  by 
granular,  refractive  points  of  irregular  shape  and  size. 
These  eventually  coalesce  and  leave  the  remainder  of 
the  cell  clear  and  transparent.  When  this  coalescence 
of  highly  refractive  particles  is  complete  the  spore  is  per- 
fected. In  appearance,  the  spore  is  oval  or  round,  very 
highly  refractive,  and  of  a  glistening  appearance.  It  is 


SPORE-FORMATION.  35 

easily  differentiated  from  the  remainder  of  the  cell, 
which  now  consists  only  of  a  cell-membrane  and  a  per- 
fectly transparent,  clear  fluid  which  surrounds  the  spore. 
Eventually  both  the  cell-membrane  and  its  fluid  con- 
tents disappear,  leaving  the  oval  spore  free  in  the  medium 
in  which  it  has  been  formed. 

The  spore,  when  perfectly  developed,  is  highly  glis- 
tening, oval  in  contour,  and  has  the  appearance  of  being 
surrounded  by  a  dark,  sharply  defined  border.  It  pos- 
sesses no  motion  other  than  the  mechanical  tremor  com- 
mon to  all  insoluble  microscopic  particles  suspended  in 
fluids,  and  it  remains  quiescent  until  conditions  favor- 
able to  its  subsequent  development  into  the  vegetative 
form  from  which  it  originated,  appear.  Occasionally 
the  membrane  of  the  vegetative  cell  in  which  the  spore 
is  formed  does  not  disappear  from  around  it,  and  the 
spore  may  then  be  seen  lying  in  a  very  delicate  tubular 
envelope.  Now  and  then  remnants  of  the  envelope  may 
be  noticed  adhering  to  the  spore  which  has  not  yet  be- 
come completely  free. 

When  stained,  the  spore-containing  cells  do  not  take 
up  the  dyes  in  a  homogeneous  way.  By  the  ordinary 
methods  the  spores  do  not  stain,  so  that  they  appear  in 
the  stained  cells  as  pale,  transparent,  oval  bodies,  sur- 
rounded by  the  remainder  of  the  cell,  which  has  taken 
up  the  staining. 

A  single  cell  produces  but  one  spore.  This  may  be 
located  either  at  an  extremity  or  in  the  centre  of  the 
cell. 

Occasionally  spore-formation  is  accompanied  by  an 
enlargement  of  the  cell  at  the  point  at  which  the  process 
is  in  progress.  As  a  result,  the  outline  of  the  cell  loses 
its  regular  rod  shape  and  becomes  that  of  a  club,  a 


•°>6  BACTERIOLOGY. 

drum-stick,  or  a  lozenge,  depending  upon  whether  the 
location  of  the  spore  is  to  be  at  the  pole  or  in  the  centre 
of  the  cell. 

In  addition  to  the  property  of  spore-formation  there  is 
another  striking  difference  between  the  members  of  the 
rod-shaped  organisms,  namely,  the  property  of  motility 
which  many  of  them  are  seen  to  possess.  This  power  of 
motion  is  due  to  the  possession  by  the  motile  bacilli 
of  very  delicate,  hair-like  appendages  or  flagellae,  by  the 
lashing  motions  of  which  the  rods  possessing  them  are 
propelled  through  the  fluid.  In  some  cases  the  flagella? 
come  off  from  but  one  end  of  a  bacillus,  either  singly  or 
in  a  bunch  ;  again,  they  may  be  seen  at  both  poles,  and 
in  some  cases,  especially  with  the  bacillus  of  typhoid 
fever,  they  are  given  off  from  the  whole  surface  of  the 
rod. 

For  a  long  time  the  motility  of  certain  of  the  bacteria 
was  supposed  to  be  due  to  the  possession  of  some  such 
form  of  locomotive  apparatus  because  similar  appendages 
had  been  seen  in  certain  of  the  large  motile  spirilla  found 
in  stagnant  water,  but  it  was  not  until  very  recently 
that  the  accuracy  of  this  suspicion  was  actually  demon- 
strated. By  a  special  method  of  staining,  Loffler  has 
been  able,  in  a  number  of  cases,  to  render  visible  these 
hair-like  appendages.  His  method  consists  in  the  em- 
ployment of  a  mordant,  by  the  aid  of  which  the  flagelke 
are  caused  to  retain  the  staining,  and  thus  become 
visible.  Loffler's  method  of  staining  will  be  found  in 
the  chapter  devoted  to  this  part  of  the  technique. 


CHAPTER    III. 

Principles  of  sterilization  by  heat — Different  methods  employed — 
Principles  of  discontinued  sterilization — Sterilization  under  pressure 
— Apparatus  employed. 

BY  the  term  sterilization,  as  employed  here,  is  under- 
stood the  destruction  of  bacteria  by  heat.  It  is  accom- 
plished in  two  ways  :  either  by  dry  heat,  or  by  moist 
heat  in  the  form  of  steam. 

Experiments  have  taught  us  that  the  process  of  steril- 
ization by  dry  heat  has  a  relatively  limited  application 
because  of  its  many  disadvantages.  For  successful  ster- 
ilization by  the  method  of  dry  heat,  not  only  is  a  rela- 
tively high  temperature  essential,  but  the  substances 
under  treatment  must  be  exposed  to  this  temperature  for 
a  comparatively  long  time.  Its  penetrating  action  into 
the  substances  which  are  to  be  sterilized  is,  moreover, 
much  less  energetic  than  that  of  steam.  Many  substances 
of  vegetable  and  animal  origin  are  rendered  useless  by 
subjection  to  the  dry  method  of  sterilization.  For  these 
reasons  there  are  comparatively  few  substances  which 
may  be  sterilized  in  this  way  without  serious  damage  to 
their  further  usefulness. 

Successful  sterilization  by  dry  heat  cannot  usually  be 
accomplished  at  a  temperature  lower  than  150°  C., 
and  to  this  degree  of  heat  must  the  objects  be  subjected 
for  not  less  than  one  hour.  For  the  sterilization, 
therefore,  of  the  organic  materials  of  which  the  media 
employed  in  bacteriological  work  are  composed,  and 
of  domestic  articles,  such  as  cotton,  woollen,  wooden,' 

3 


:>S  BACTERIOLOGY. 

and  leather  articles,  this  method  is  entirely  impracticable. 
In  bacteriological  work  its  application  is  limited  to  the 
sterilization  of  glass-ware  principally — such,  for  exam- 
ple, as  flasks,  plates,  small  dishes,  test-tubes,  pipettes — 
and  such  metal  instruments  as  are  not  seriously  injured 
by  the  high  temperature. 

With  sterilization  by  moist  heat — steam — the  condi- 
tions are  much  more  favorable.  The  penetrating  action 
of  the  steam  is  not  only  much  more  energetic,  but  the 
temperature  at  which  this  is  ordinarily  accomplished  is, 
as  a  rule,  not  destructive  in  its  action.  This  is  conspic- 
uously seen  in  the  work  of  the  laboratory.  The  culture 
media,  composed  in  the  main  of  decomposable  organic 
materials,  which  would  be  rendered  entirely  worthless  if 
exposed  to  the  dry  method  of  sterilization,  sustain  no 
injury  whatever  when  intelligently  subjected  to  an  equally 
effective  sterilization  with  steam.  The  same  may  be  said 
of  cotton  and  woollen  fabrics,  bedding,  clothing,  etc. 

Aside  from  the  relations  of  the  two  methods  to  the 
materials  to  he  sterilized,  their  action  toward  the  organ- 
isms to  be  destroyed  is  quite  different.  The  penetrating 
action  of  the  steam  renders  it  by  far  the  more  efficient 
agent  of  the  two.  The  spores  of  several  organisms  which 
are  killed  by  an  exposure  of  but  a  few  moments  to  the 
action  of  steam,  resist  the  destructive  action  of  dry  heat 
at  a  higher  temperature  for  a  much  greater  length  of 
time. 

These  differences  will  be  strikingly  brought  out  in 
the  experimental  work  on  this  subject.  For  our  purposes 
it  is  necessary  to  say  that  the  two  methods  have  the  fol- 
lowing applications : 

The  dry  method,  at  a  temperature  of  150°-180°  C., 
for  one  hour,  is  employed  for  the  sterilization  of  glass- 


PRINCIPLES  OF  STERILIZATION    BY  HEAT.       39 

ware :  flasks,  test-tubes,  culture  dishes,  pipettes,  plates, 
etc. 

The  sterilization  by  steam  is  practised  with  all  media, 
whether  fluid  or  solid.  Bouillon,  milk,  gelatin,  agar- 
agar,  potato,  etc.,  are  under  no  conditions  to  be  subjected 
to  the  dry  heat. 

The  methods  by  which  heat  is  employed  in  processes 
of  sterilization  vary  with  circumstances.  In  its  em- 
ployment as  dry  heat  its  application  is  always  continuous 
— i.  e.j  the  objects  to  be  sterilized  are  simply  exposed  to 
the  proper  temperature  for  the  length  of  time  necessary 
to  destroy  all  living  organisms  which  may  be  upon  them. 
With  steam,  on  the  other  hand,  the  objects  to  be  steril- 
ized are  frequently  of  such  a  nature  that  a  prolonged 
application  of  the  heat  would  materially  injure  them. 
For  this  reason  steam  is  usually  applied  intermittently 
and  for  short  periods  of  time.  The  principles  involved 
in  this  method  of  sterilization  depend  upon  differences 
of  resistance  toward  heat  which  the  organisms  to  be 
destroyed  are  seen  to  possess  at  different  stages  of 
their  development.  During  the  life  history  of  many  of 
the  bacilli  there  is  a  time  in  which  the  resistance  of  the 
organism  toward  the  action  of  both  chemical  and  thermal 
agents  is  much  higher  than  at  other  stages  of  its  devel- 
opment. This  increased  power  of  resistance  is  seen  to 
exist  when  these  organisms  are  in  the  spore  or  resting 
stage,  to  which  reference  has  already  been  made. 
When  in  the  vegetative  or  growing  stage,  most  of 
these  organism  are  killed  in  a  short  time  by  a  relatively 
low  temperature,  whereas,  when  conditions  have  arisen 
which  favor  the  production  of  spores,  these  spores  are 
seen  to  be  capable  of  resisting  very  much  higher  tem- 
peratures for  an  appreciably  longer  time.  These  differ- 


40  BACTERIOLOGY. 

ences  in  resistance  toward  heat  which  the  spore-forming 
organisms  are  seen  to  possess  at  their  different  stages  of 
development,  are  taken  advantage  of  in  the  process  of 
sterilization  by  steam  which  is  known  as  the  fractional 
or  intermittent  method,  and  form  the  principle  on  which 
the  method  is  based. 

The  object  aimed  at  in  this  method  is  to  destroy  the 
organisms  in  the  shortest  time  and  with  the  least  amount 
of  heat.  It  is  accomplished  by  subjecting  them  to  the 
elevated  temperature  at  the  time  when  they  are  in  the 
vegetating  or  growing  stage — i.  e.,  the  stage  at  which 
they  are  least  resistant.  In  order  to  accomplish  this  it 
is  necessary  that  there  should  exist  conditions  of  tem- 
perature, nutrition,  and  moisture  which  favor  the  vege- 
tation of  the  bacilli,  and  the  germination  of  any  spores 
that  may  be  present.  When  these  surroundings  aiv 
found,  the  spore-forming  organisms  are  not  only  less 
likely  to  go  into  the  spore  stage  than  when  their  en- 
vironments .are  less  favorable  to  their  vegetation,  but 
spores  which  may  already  exist  develop  very  quickly 
into  mature  cells. 

It  is  plain,  then,  that  with  the  first  application  of  the 
steam  to  the  substance  to  be  sterilized,  the  mature  vege- 
tative forms  of  these  organisms  are  destroyed,  while  cer- 
tain spores  which  might  have  been  present  resist  this 
treatment,  providing  the  sterilization  has  not  been  con- 
tinued for  too  long  a  time.  If  now  the  sterilization  is 
discontinued,  and  the  material  which  presents  conditions 
favorable  to  the  germination  of  the  spores  is  allowed  to 
stand  for  a  time,  usually  for  about  twenty-four  hours, 
at  a  temperature  of  from  30°-35°  C.,  those  spores  which 
resisted  the  action  of  the  steam  will  in  the  course  of  this 
time  germinate  into  the  less  resistant  vegetative  cells- 


STERILIZATION.  41 

A  second  short  exposure  to  the  steam  kills  these  forms 
in  turn,  and  by  a  repetition  of  this  process  all  organisms 
which  were  present  may  be  destroyed  without  the  appli- 
cation of  the  steam  having  been  at  any  time  of  long 
duration.  In  this  process  the  usual  plan  is  to  subject 
the  materials  to  be  sterilized  to  the  action  of  steam, 
under  the  normal  conditions  of  temperature  and  pressure, 
for  fifteen  minutes  on  each  of  three  successive  days,  and 
during  the  intervening  days  to  retain  them  at  a  temper- 
ature of  about  25°-30°  C.  At  the  end  of  this  time  all 
living  organisms  which  were  present  will  have  been  de- 
stroyed, and  unless  opportunity  is  given  for  the  access  of 
new  organisms  from  without,  the  substances  thus  treated 
remain  sterile. 

It  must  be  borne  in  mind  that  this  method  of  steriliza- 
tion  is  only  applicable  in  those  cases  which  present 
conditions  favorable  to  the  germination  of  the  spores 
into  mature  vegetative  cells.  Dry  substances  or  organic 
materials  in  which  decomposition  is  far  advanced,  where 
the  proper  conditions  for  the  germination  of  spores  are 
not  present,  cannot  be  successfully  sterilized  by  the 
intermittent  method. 

The  process  of  fractional  sterilization  at  low  tempera- 
tures is  based  upon  exactly  the  same  principle,  but  dif- 
fers in  two  respects,  viz.,  it  requires  a  longer  time  for 
its  accomplishment,  and  the  temperature  at  which  it  is 
conducted  is  not  raised  above  68°-70°  C.  It  is  em- 
ployed for  the  sterilization  of  easily  decomposable  mate- 
rials, which  would  be  rendered  useless  by  the  tempera- 
ture of  steam,  but  which  remain  intact  at  the  temperature 
employed.  This  process  requires  that  the  material  to  be 
sterilized  should  be  subjected  to  a  temperature  of  68°- 
-70°'-  (2.  for  6lfev  hour  on  each  of  six  successive  days,  an 


42  BACTERIOLOGY. 

interval  of  twenty-four  hours  being  allowed  between  the 
exposures  to  this  temperature  for  the  germination  of 
spores  into  mature  cells.  During  this  interval  the  sub- 
stances under  treatment  are  kept  at  about  25°-30°  C. 
The  temperature  employed  in  this  process  suffices  to 
destroy  the  vitality  of  almost  all  organisms  in  the  vege- 
tative stage  in  about  one  hour.  Blood-serum  is  always 
sterilized  by  the  intermittent  method  at  low  temperature. 

Sterilization  by  steam  is  also  practised  by  what  may 
be  called  the  direct  method.  That  is  to  say,  both  the 
mature  organisms  and  the  spores  which  may  be  present 
in  the  material  to  be  sterilized  are  destroyed  by  a  single 
exposure  to  the  steam.  In  this  method  steam  at  its 
ordinary  temperature  and  pressure — live  steam  or  stream- 
ing steam  as  it  is  called — is  employed  just  as  in  the  first 
method  described,  but  it  is  allowed  to  act  for  a  much 
longer  time,  usually  not  less  than  one  hour.  Or,  steam 
under  pressure,  and  consequently  of  a  higher  tempera- 
ture, is  now  frequently  employed.  In  this  method  a 
single  exposure  of  fifteen  minutes  is  sufficient  for  the 
destruction  of  all  bacilli  and  their  spores,  providing  the 
pressure  of  the  steam  is  not  less  than  one  atmosphere 
over  and  above  that  of  normal — this  is  equivalent  to 
an  approximate  temperature  of  122°  C.  to  which  the 
organisms  are  exposed. 

The  objection  to  both  of  these  methods  of  direct 
sterilization  by  steam  is  that  many  substances  which  it 
is  desirable  to  retain  in  as  near  their  normal  condition 
as  possible  are  materially  altered  by  this  energetic  form 
of  treatment.  Gelatin  is  not  only  rendered  cloudy,  but 
loses  the  power  of  gelatinzing.  Many  of  the  other 
media  contain  always  a  fine  precipitate  after  this  method  ; 
in  fact,  for  most  of  the  media  which  we  employ,  the 


STERILIZATION    APPARATUS. 


43 


discontinued  method  at  the  temperature  of  streaming 
steam  gives  the  most  satisfactory  results. 

For  sterilization  by  steam  the  apparatus  commonly 
employed  has  until  recently  been  the  cylindrical  boiler 
recommended  by  Koch,  a  cut  of  which  may  be  seen  in 
Fig.  1. 


FK;.  1. 


Its  construction  is  very  simple.  It  consists  of  a 
copper  cylinder,  the  lower  fourth  of  which  is  somewhat 
larger  in  diameter  than  the  remaining  three- fourths,  and 
acts  as  a  reservoir  for  the  water  from  which  the  steam 
is  to  be  generated.  Covering  this  section  of  the  cylinder 
is  a  wire  rack  or  grating  through  which  the  steam 
passes,  and  which  serves  as  a  bottom  upon  which  the 


14  BACTERIOLOGY. 

materials  to  be  sterilized  rest.  Above  this,  comprising 
the  remaining  three-fourths  of  the  cylinder,  is  the  cham- 
ber for  the  reception  of  the  materials  over  and  through 
which  the  steam  is  to  pass.  The  cylinder  is  closed  by  a 
snugly-fitting  cover  through  which  are  usually  two  per- 
forations into  which  a  thermometer  and  a  manometer  may 
be  inserted.  The  whole  of  the  outer  surface  of  the  appa- 
ratus is  encased  in  a  non-conducting  mantle  of  asbestos 
or  felt. 

The  water  is  heated  by  a  gas-flame  placed  in  an  en- 
closed chamber,  upon  which  the  apparatus  rests,  vvhieh 
serves  to  diminish  the  loss  of  heat  and  deflection  of  the 
flame  through  the  action  of  draughts.  The  apparatus 
is  simple  in  construction,  and  the  only  point  which  is  to 
be  observed  while  using  it  is  the  level  of  the  water  in 
the  reservoir.  On  the  reservoir  is  a  water-gauge  which 
indicates  at  all  times  the  amount  of  water  in  the  appa- 
ratus. The  amount  of  water  should  never  be  too  small  to 
be  indicated  by  the  gauge,  otherwise  there  is  danger  of 
the  reservoir  becoming  dry  and  the  bottom  of  the  appa- 
ratus being  destroyed  by  the  direct  action  of  the  flame. 

A  sterilizer  now  gaining  favor  for  use  in  laboratories 
is  an  apparatus  originally  intended  for  use  in  the  kitchen. 
It  is  the  so-called  "Arnold  Steam  Sterilizer."  It  is  very 
ingenious  in  its  construction  as  well  as  economical  in  its 
employment. 

The  difference  between  this  apparatus  and  that  just 
described  is  that  it  provides  for  the  condensation  of  the 
steam  after  its  escape  from  the  sterilizing  chamber,  and 
returns  the  water  of  condensation  automatically  to  the 
reservoir,  so  that  in  practice  the  apparatus  requires  but 
little  attention,  as  with  moderate  care  there  is  no  fear  of 


STERILIZATION    UNDER    PRESSURE.          45 

the  water  in  the  reservoir  becoming  exhausted  and  the 
consequent  destruction  of  the  sterilizer. 

Fig.  2  gives  an  illustration  of  this  apparatus. 

FIG.  2. 


For  sterilization  by  steam  under  pressure  several 
special  forms  of  apparatus  exist.  The  principles  of 
them  all  are,  however,  the  same.  They  provide  for  the 
generation  of  steam  in  a  chamber  from  which  it  cannot 
escape  when  the  apparatus  is  closed.  Upon  the  cover  of 
this  chamber  is  a  safety-valve,  which  can  be  regulated  so 
that  any  degree  of  pressure  desirable  can  be  maintained 
within  the  sterilizing  chamber.  These  sterilizers  are 
known  as  "  digestors "  and  also  by  the  French  name 
"autoclav.''  Their  construction  can  best  be  understood 
by  reference  to  Fig.  3. 

The  dry  sterilizers  used  in  laboratories  arc  simply 
double- walled  boxes  of  Russian  or  Swedish  iron  (Fig.  4), 

a* 


46 


BACTERIOLOGY 
FIG.  3. 


having  a  double-walled  door,  which  closes  tightly,  and 
a  heavy  copper  bottom.  They  are  arranged  with  venti- 
lating openings  for  the  escape  of  the  contained  air  and 
the  entrance  of  the  heated  air.  The  flame,  usually  from 
a  rose  burner  (Fig.  5),  is  applied  directly  to  the  bottom. 
The  heat  circulates  from  the  lower  surface  around  about 
the  apparatus  through  the  space  between  its  walls. 

The  construction  of  the  copper  bottom  of  the  appa- 
ratus upon  which  the  flame  impinges,  is  designed  to 
prevent  the  direct  action  of  the  flame  upon  the  sli.<i- 


ORDINARY    DRY    STERILIZER. 


47 


iron  bottom  of  the  chamber.  It  consists  of  several 
copper  plates  placed  one  above  the  other,  but  with  a  space 
of  about  4  to  5  mm.  between  the  plates.  These  copper 
bottoms  after  a  time  become  burned  out,  and  unless  they 
are  replaced  the  apparatus  is  useless.  The  older  form 

FIG.  4. 


FIG.  5. 


of  sterilizers  are  so  constructed  that  their  repair  is  a 
matter  involving  some  time  and  expense.  To  meet  this 
objection  I  have  had  constructed  a  sterilizer  in  all  re- 
spects similar  to  the  old  form  except  in  the  arrangement 
of  this  copper  bottom.  This  is  so  made  that  it  can  b£ 
easily  slipped  in  and  out,  so  that  by  keeping  several  sets 
of  copper  plates  on  hand  a  new  one  can  readily  be 
slipped  into  the  apparatus  when  the  old  one  is  burned 
out. 

In  the  employment  of  the  dry  sterilizer  care  should 


48  BACTERIOLOGY. 

always  be  given  to  the  condition  of  the  copper  bottom  ; 
for  the  direct  application  of  the  heat  to  the  sheet-iron 
plate  upon  which  the  substances  to  be  sterilized  stand, 
results  not  only  in  destruction  of  the  apparatus,  but 
frequently  in  destruction  of  the  substances  undergoing 
sterilization. 

Since  the  temperature  at  which  this  form  of  steriliza- 
tion is  usually  accomplished  is  high — 150°  to  180°  C. 
— it  is  well  to  have  the  apparatus  encased  in  asbestos 
boards,  to  diminish  the  radiation  of  heat  from  its  sur- 
faces. This  not  only  confines  the  heat  to  the  apparatus, 
but  guards  against  the  destructive  action  of  the  radiated 
heat  on  woodwork,  furniture,  etc.,  that  may  be  in  the 
neighborhood. 


CHAPTER    IV. 

Disinfection — Antiseptics — Inorganic  salts  as  disinfectants — The 
value  of  corrosive  sublimate — Heat. 

IN  contradistinction  to  sterilization,  disinfection  im- 
plies the  destruction  of  bacteria  by  chemical  processes. 
In  the  destruction  of  bacteria  by  means  of  chemical 
substances,  there  occurs  most  probably  a  definite  chemical 
reaction ;  that  is  to  say,  the  characteristics  of  both  the 
bacteria  and  the  agent  employed  in  their  destruction  are 
lost  in  the  production  of  a  third  body,  the  result  of  their 
combination.  It  is  impossible  to  say  with  absolute  cer- 
tainty, as  yet,  that  this  is  the  case,  but  the  evidence  that 
is  rapidly  accruing  from  the  more  recent  studies  upon 
disinfectants  and  their  mode  of  action  point  strongly  to 
the  accuracy  of  this  belief.  This  reaction,  in  which  the 
typical  structures  of  both  bodies  concerned  is  lost,  takes 
place  between  the  agent  employed  for  disinfection  and 
the  protoplasm  of  the  bacteria.  For  example,  in  the 
reaction  that  is  seen  to  take  place  between  the  salts 
of  mercury  and  albuminous  bodies  there  results  a  third 
compound,  which  has  neither  the  characteristics  of  mer- 
cury nor  of  albumin,  but  partakes  of  the  peculiarities 
of  both ;  it  is  a  combination  of  albumin  and  mercury 
known  by  the  indefinite  term  "  albuminate  of  mercury." 
Some  such  reaction  as  this  occurs  when  the  soluble 
salts  of  mercury  are  brought  in  contact  with  bacteria. 
This  view  has  recently  been  strengthened  by  the  experi- 
ments of  Geppert,  in  which  the  reaction  was  caused  to 


50  BACTERIOLOGY. 

take  place  between  the  spores  of  the  anthrax  bacillus 
and  a  solution  of  mercuric  chloride,  the  result  being 
the  apparent  destruction  of  the  living  properties  of  the 
spores  by  the  formation  of  this  third  compound.  In 
these  experiments  it  was  shown  that  though  this  com- 
bination had  taken  place,  still  it  did  not  of  necessity 
imply  the  complete  death  of  the  protoplasm  of  the 
spores,  for  if  by  proper  means  the  combination  of  mer- 
cury with  their  protoplasm  was  broken  up,  many  of 
the  spores  returned  from  their  condition  of  apparent 
death  to  that  of  life,  with  all  their  previous  disease- 
producing  and  cultural  peculiarities.  Geppert  employed 
a  solution  of  ammonium  sulphide  for  the  purpose  of 
destroying  the  combination  of  spore-protoplasm  and 
mercury.  The  mercury  was  precipitated  from  the  pro- 
toplasm as  an  insoluble  sulphide,  and  the  protoplasm 
of  the  spores  returned  to  its  original  condition.  These 
and  other  somewhat  similar  experiments  have  given  an 
entirely  new  impulse  to  the  study  of  disinfectants, 
and  in  the  light  shed  by  them  many  of  our  previ- 
ously formed  ideas  concerning  the  action  of  disinfecting 
agents  must  be  modified.  The  process  is  not  a  cata- 
lytic one — i.e.,  occurring  simply  as  a  result  of  the 
presence  of  the  disinfecting  body  which  is  not  itself 
destroyed  in  its  process  of  destruction — but  is,  as  said,  a 
definite  chemical  reaction  which  takes  place  within  cer- 
tain more  or  less  fixed  limits  ;  that  is  to  say,  with  a  given 
amount  of 'the  disinfectant  employed,  just  so  much  work, 
expressed  in  terms  of  disinfection — destruction  of  bac- 
teria— can  be  accomplished. 

Another  point  in  favor  of  this  view  is  the  increased 
energy  of  the  reaction  with  elevation  of  temperature. 
Just  as  in  many  other  chemical  phenomena,  the  intensity 


DISINFECTION    AND    ANTISEPTICS.  51 

of  the  reaction  becomes  greater  under  the  influence  of 
heat,  so  in  the  process  of  disinfection  the  combination 
between  the  disinfectant  and  the  organisms  to  be  de- 
stroyed is  much  more  energetic  at  a  temperature  of  37° 
to  39°  C.  than  it  is  at  12°  to  15°  C. 

What  has  been  said  refers  more  particularly  to  the 
inorganic  salts  which  are  employed  for  this  purpose.  It 
is  probable  that  the  organic  bodies  which  possess  disin- 
fectant properties  owe  this  power  to  some,  such  similar 
reaction,  though,  as  yet,  these  substances  have  not  been 
so  thoroughly  studied  in  this  relation. 

The  reaction  between  these  inorganic  salts  and  albu- 
minous bodies  is  not  a  selective  action ;  they  combine 
in  most  instances  with  any  or  all  protoplasmic  bodies 
present.  For  this  reason  the  efficacy  of  the  practical 
application  of  many  of  the  commonly  employed  dis- 
infectants is  a  matter  of  grave  doubt.  For  example, 
the  disinfection  of  excreta,  sputum,  or  blood  containing 
pathogenic  organisms,  by  means  of  corrosive  sublimate, 
is  a  procedure  of  very  questionable  success.  The  amount 
of  sublimate  employed  may  be  entirely  used  up  and 
rendered  inactive  as  a  disinfectant  by  the  ordinary 
protoplasmic  substances  present  without  having  any 
appreciable  effect  upon  the  bacteria  which  may  be  in  the 
mass. 

These  remarks  are  introduced  in  order  to  guard 
against  the  implicit  confidence  so  often  placed  in  the 
disinfecting  value  of  corrosive  sublimate.  lu  bacterio- 
logical laboratories,  where  there  is  constantly,  more  or 
less  of  infectious  material,  it  is  the  custom,  with  few 
exceptions,  to  have  vessels  containing  solutions  of  corro- 
sive sublimate  at  hand,  by  which  infectious  materials 
may  be  rendered  harmless.  The  value  of  this  procedure, 


52  HEAT. 

as  we  have  just  learned,  is  always  more  or  less  question- 
able, especially  in  those  cases  in  which  the  substance  to 
be  disinfected  is  of  an  albuminous  nature.  With  the  in- 
troduction of  such  substances  into  the  sublimate  solution 
the  mercury  is  quickly  precipitated  by  the  albumin  and 
its  disinfecting  properties  may  be  entirely  destroyed ; 
we  may  in  a  very  short  time  have  little  else  than  water 
containing  a  precipitate  of  albumin  and  mercury,  in  so 
far  as  its  value  as  a  disinfectant  is  concerned. 

In  the  laboratory,  then,  heat  is  the  surest  agent  to 
employ.  All  tissues  containing  infectious  organisms 
should  be  burned,  and  all  cloths,  test-tubes,  flasks,  and 
dishes  should  be  boiled  in  2  per  cent,  soda  solution  for 
fifteen  to  twenty  minutes,  or  placed  in  the  steam  sterilizer 
for  at  least  half  an  hour. 

Intestinal  evacuations  may  best  be  disinfected  with 
milk  of  lime,  a  mixture  composed  of  lime  in  solution  and 
in  suspension.  This  should  be  thoroughly  mixed  with 
the  evacuations  until  the  mass  reacts  distinctly  alkaline, 
and  should  remain  in  contact  with  the  infective  sub- 
stance for  several  hours. 

Sputum  in  which  tubercle  bacilli  are  present,  as  well 
as  the  vessel  containing  it,  must  be  boiled  in  soda  solu- 
tion for  fifteen  minutes  or  steamed  in  the  sterilizer  for 
at  least  half  an  hour. 

On  the  whole,  for  the  laboratory  we  should  as  yet 
rely  more  upon  the  destructive  properties  of  heat  than 
upon  that  of  chemical  agents. 

From  what  has  been  said,  the  absurdity  of  sprinkling 
about,  here  and  there,  a  little  carbolic  acid  or  in  placing 
about  apartments  in  which  infectious  diseases  are  in 
progress  little  vessels  of  carbolic  acid,  must  be  plain. 
The  disinfection  of  water-closets  and  cesspools  by  allow- 


DISINFECTANT    AGENTS.  53 

ing  now  and  then  a  few  cubic  centimetres  of  some  so- 
called  disinfectant  to  trickle  through  the  pipes  is  a 
failure.  A  disinfectant  must  be  applied  to  the  bacteria, 
and  must  be  in  contact  with  them  for  a  long  enough  time 
to  insure  the  destruction  of  their  life.  In  the  light  of  the 
latest  experiments  upon  disinfectants,  the  place  formerly 
occupied  by  many  agents  in  the  list  of  substances  em- 
ployed for  the  purpose  will  most  likely  be  changed  as 
they  are  studied  more  closely. 

The  agents,  then,  which  will  prove  of  most  value  in 
the  laboratory  for  the  purpose  of  rendering  infectious 
materials  harmless  are  :  Heat,  either  by  burning,  by 
steaming  for  from  half  an  hour  to  an  hour,  or  by  boiling 
in  a  2  per  cent,  soda  solution  for  fifteen  minutes ;  a  solu- 
tion of  chlorinated  lime  ("  chloride  of  lime  "),  in  which 
the  percentage  of  chlorine  is  high  ;  and  milk  of  lime. 
The  materials  to  be  disinfected  in  either  of  the  lime 
solutions  should  remain  in  them  for  several  hours.  The 
solutions  should  be  freshly  prepared  when  needed,  as 
they  rapidly  decompose  upon  standing. 

Antiseptic.  An  antiseptic  is  a  body  which,  by  its 
presence,  prevents  the  growth  of  bacteria  without  of 
necessity  killing  them.  A  body  may  be  an  antiseptic 
without  possessing  disinfecting  properties  to  any  very 
high  degree,  but  a  disinfectant  is  always  an  antiseptic 
as  well. 


CHAPTER    V. 

The  principles  involved  in  the  methods  of  isolation  of  bacteria  in 
pure  culture  by  the  plate  method  of  Koch — Materials  employed. 

SINCE  the  introduction  of  the  plate  method  for  iso- 
lating in  pure  culture  the  individual  species  from  mix- 
tures of  bacteria,  a  number  of  modifications  have  been 
adopted,  but  the  principle  of  them  all  is  the  same. 
The  observation  which  led  to  their  development  was 
a  very  simple  one,  and  one  that  is  commonly  before 
us.  Koch  noticed  that  on  solid  substances,  such,  tor 
example,  as  a  slice  of  potato,  which  had  been  exposed 
for  a  time  to  the  air  and  which  afforded  proper  nourish- 
ment for  the  lower  organisms,  there  developed  after  a 
short  time  small  patches  of  material  which  proved  to  be 
colonies  of  bacteria.  Each  ofthe.se  colonies  on  closer  ex- 
amination showed  itself  to  be,  as  a  rule,  composed  of  but 
a  single  species.  There  was  no  tendency  toward  a  con- 
fluence of  these  colonies,  and  from  the  differences  in 
their  naked-eye  appearances,  it  was  easy  to  see  that  they 
were  mostly  the  outgrowth  of  different  species  of  bacteria. 

The  question  that  then  presented  itself  was :  If  from 
a  mixture  of  organisms  floating  in  the  air  it  is  possible 
in  this  way  to  obtain  in  pure  cultures  the  individual 
organisms  composing  the  mixture,  what  means  can  be 
employed  for  obtaining  the  same  results  at  will  from 
mixtures  of  different  organisms  when  found  under  other 
conditions  ? 


PLATE    METHOD    OF    KOCH.  55 

It  was  plain  that  the  organisms  were  to  be  distin- 
guished, the  one  from  the  other,  only  by  the  structure 
and  general  appearance  of  the  colonies  growing  from 
them,  for  upon  their  morphology  alone  this  is  impossible. 

What  means  could  be  devised,  then,  for  separating  the 
individual  members  of  a  mixture,  in  such  a  way  that 
they  would  remain  in  a  fixed  position,  and  be  sufficiently 
widely  separated,  the  one  from  the  other,  as  not  to  inter- 
tere  with  the  production  of  colonies  of  characteristic 
appearance,  which  would,  under  the  proper  conditions, 
develop  from  each  individual  cell  ? 

If  a  test-tube  of  decomposed  bouillon  were  poured  out 
upon  a  large  flat  surface,  the  individual  bacteria  in  the 
mass  would  be  very  much  more  widely  separated  the; 
one  from  the  other  than  they  were  when  the  bouillon 
was  in  the  tube.  But  they  are  in  a  fluid  medium,  and 
there  is  no  possibility  of  their  either  remaining  separated 
or  of  their  forming  colonies  under  these  conditions,  so 
that  it  is  impossible  by  this  means  to  pick  out  the  in- 
dividuals in  the  mixture. 

If,  however,  it  is  possible  to  find  some  substance 
which  possesses  the  property  of  being  at  one  time  fluid 
and  at  another  time  solid,  which  can  be  added  to  this 
bouillon  without  in  any  way  interfering  with  the  life 
functions  of  the  bacteria,  then,  as  solidification  sets  in, 
the  organisms  will  be  fixed  in  their  positions  and  the 
conditions  will  be  analogous  to  that  seen  on  the  bit  of 
potato. 

Gelatin  possesses  this  property.  At  a  temperature 
which  does  not  interfere  with  the  life  of  the  organisms 
it  is  quite  fluid,  whereas  when  subjected  to  a  lower 
temperature  it  solidifies.  When  once  solid  it  may  be 


56  BACTERIOLOGY. 

kept  at  a  temperature  favorable   to  the  growth  of  the 
bacteria  and  retain  its  solid  condition. 

Gelatin  was  added  to  the  fluids  containing  mixtures 
of  bacteria,  and  the  whole  was  then  poured  upon  a  hir^v 
flat  surface,  allowed  to  solidify,  and  the  results  noted. 
It  was  found  that  the  conditions  seen  on  the  slice  of 
potato  could  be  reproduced,  that  the  individuals  in  the 
mixture  of  bacteria  grew  well  in  the  gelatin,  and,  as  on  the 
potato,  grew  in  colonies  of  typical  macroscopic  structure, 
so  that  they  could  easily  be  separated  the  one  from  the 
other  by  their  naked-eye  appearances.  It  was  necessary, 
however,  to  use  a  more  dilute  mixture  of  bacteria  than 
that  seen  in  the  original  decomposed  bouillon.  The 
number  of  individuals  in  the  tube  was  so  enormous 
that  on  the  gelatin  plate  they  were  so  closely  packed 
together  that  it  was  not  only  impossible  to  pick  them 
out  because  of  their  proximity  the  one  to  the  other,  but 
also  because  this  close  packing  together  materially  inter- 
fered with  the  production  of  those  characters  by  means 
of  which  differences  can  be  seen  with  the  naked  eye. 
The  numbers  of  organisms  were  then  diminished  by  a  pro- 
cess of  dilution,  consisting  of  transferring  a  small  portion 
of  the  original  mixture  into  a  second  tube  of  sterilized 
bouillon  to  which  gelatin  had  been  added  and  liquefied  ; 
from  this  a  similar  portion  was  added  to  a  third  galatin- 
bouillon  tube,  and  so  on.  These  were  then  poured  upon 
large  surfaces  and  allowed  to  solidify.  The  results  were 
entirely  satisfactory.  On  the  gelatin,  plates  from  t lie- 
original  tube,  as  was  expected,  the  colonies  were  too 
numerous  to  be  of  any  use;  on  the  plates  made  from 
the  first  dilution  they  were  much  fewer  in  number,  but 
still  they  were  usually  too  numerous  and  too  closely 
packed  to  permit  of  characteristic  growth  ;  but  on  the 


CULTURE  MATERIALS  EMPLOYED.     57 

second  dilution  they  were,  as  a  rule,  fewer  in  number 
and  widely  separated,  so  that  the  individuals  of  each 
species  were  in  no  way  prevented  by  the  proximity  of  its 
neighbors  from  growing  in  its  own  typical  way.  There 
was  then  no  difficulty  in  picking  out  the  colonies  result- 
ing from  the  growth  of  the  different  individual  bacteria. 

Such,  then,  are  the  principles  upon  which  Koch's 
method  for  the  isolation  of  bacteria  by  means  of  solid 
media  is  based. 

The  fundamental  part  of  the  media  employed  is  the 
bouillon,  which  contains  all  the  elements  necessary  for 
the  nutrition  of  most  bacteria,  the  gelatin  being  em- 
ployed limply  for  the  purpose  of  rendering  the  bouillon 
solid.  The  medium  on  which  the  organisms  are  growing 
is,  therefore,  simply  solidified  bouillon,  or  beef  tea. 

In  practice,  two  forms  of  gelatin  are  employed — the 
one  an  animal  or  bone  gelatin,  the  ordinary  table  gelatin  of 
good  quality;  and  the  other  a  vegetable  gelatin,  known 
as  agar-agar,  or  Japanese  gelatin,  which  is  obtained 
from  a  group  of  algae  growing  in  the  sea  along  the  coast 
of  Japan,  where  it  is  employed  as  an  article  of  diet  by 
the  natives. 

Aside  from  these  differences  in  origin  of  the  two  forms 
of  gelatin  employed,  their  behavior  toward  heat  and 
toward  bacteria  renders  them  of  different  application  in 
the  bacteriological  work.  The  animal  gelatin  liquefies  at 
a  much  lower  temperature,  and  likewise  solidifies  at  a  very 
much  lower  temperature,  than  does  the  agar-agar.  Ordi- 
nary gelatin  liquefies  at  about  24°  C.,  and  becomes  solid 
at  from  8°-10°  C.  It  may  be  employed  for  those  organ- 
isms which  do  not  require  a  higher  temperature  for  their 
development  than  22°  C.  Agar-agar,  on  the  other  hand, 
does  not  liquefy  until  the  temperature  has  reached  about 


58  BACTERIOLOGY. 

98°-99°  C.  It  remains  fluid  ordinarily  until  the  tem- 
perature has  fallen  to  38°-39°  C.,  when  it  rapidly 
solidifies.  For  our  purposes,  only  that  form  of  a«iar- 
agar  can  be  used  which  remains  fluid  at  from  38°- 
40°  C.  Agar-agar  which  remains  fluid  only  at  a  tem- 
perature above  this  point  would  be  too  hot,  when  in  a 
fluid  state,  for  use;  many  of  the  organisms  which  would 
be  introduced  into  it  would  either  be  destroyed  or 
checked  in  their  development  by  so  high  a  temperature. 
Agar-agar,  therefore,  is  for  use  in  those  cases  in  which 
the  cultivation  must  be  conducted  at  a  temperature  above 
that  at  which  gelatin  remains  solid. 

In  addition  to  the  differences  toward  temperature, 
the  relations  of  these  two  gelatins  to  bacteria  are  differ- 
ent. Many  bacteria  bring  about  alterations  in  gelatin 
which  cause  it  to  become  liquid  (probably  a  process  of 
peptonization),  in  which  state  it  remains.  There  are  no 
known  organisms  which  bring  about  such  a  change  in 
the  agar-agar. 

As  a  rule,  the  colony-formations  seen  upon  gelatine 
are  much  more  characteristic  than  those  which  develop 
on  agar-agar,  and  for  this  reason  gelatin  is  to  be  pre- 
ferred when  circumstances  will  permit.  Both  gelatin 
and  agar-agar  may  be  used  in  the  preparation  of  plates 
and  Esmarch  tubes,  subsequently  to  be  described. 


CHAPTER   VI. 

Preparation  of  media — Bouillon,  gelatin,  agar-agar,  potato,  blood- 
serum,  etc. 

As  lias  been  stated,  the  fundamental  part  of  our  cul- 
ture media  is  beef  tea,  or  bouillon. 

BOUILLON. — The  formula  of  Koch  for  the  preparation 
of  this  medium  has  undergone  many  modifications  to 
meet  special  cases,  but  for  general  use  his  original  for- 
mula is  still  retained.  It  is  as  follows :  Five  hundred 
grammes  of  finely-chopped  lean  beef,  free  from  fat  and 
tendons,  is  to  be  soaked  in  one  litre  of  water  for 
twenty-four  hours.  During  this  time  it  is  to  remain  in 
the  ice-chest  or  to  be  kept  at  a  low  temperature.  It  is 
then  to  be  strained  through  a  coarse  towel  and  pressed 
until  a  litre  of  fluid  is  obtained.  To  this  is  to  be  added 
ten  grammes  (1.0  per  cent.)  of  dried  peptone  and  five 
grammes  (0.5  per  cent.)  of  common  salt  (NaCl).  It  is 
then  to  be  rendered  exactly  neutral  or  very  slightly 
alkaline,  with  a  few  drops  of  saturated  soda  solution. 
The  flask  containing  the  mixture  is  then  to  be  placed 
either  in  the  steam  sterilizer  or  on  a  water-bath,  or  over 
a  free  flame,  and  kept  at  the  boiling-point  until  all  the 
albumin  is  coagulated,  and  the  fluid  portion  is  of  a 
clear,  pale,  straw-color.  It  is  then  filtered  through  a 
folded  paper  filter,  and  sterilized  in  the  steam  sterilizer 
by  the  fractional  method.  Certain  of  the  modifications 
of  this  method  are  of  sufficient  value  to  justify  mention. 
Most  important  is  the  neutralization.  Ordinarily,  this 


00  BACTERIOLOGY. 

is  accomplished  with  the  saturated  soda  solution,  and 
the  reaction  is  detected  with  the  ordinary  red  and  blue 
litmus  paper. 

Soda  solution  is  not  so  good  as  a  strong  solution  of 
caustic  soda  or  potash,  because  the  carbonic  acid  liberated 
from  the  sodium  carbonate  is  frequently  seen  to  give  rise 
to  a  confusing  temporary  acid  reaction  which  disappears 
on  heating.  To  obviate  this,  Schultz  ( Centralbl.  f.  Bad. 
u.  Parasitenkunde,  Bd.  x.,  Nos.  2  and  3,  1891)  recom- 
mends exact  titration  with  a  solution  of  caustic  soda. 
For  this  purpose  a  4  per  cent,  solution  of  caustic  soda  is 
prepared.  From  this  a  0.4  per  cent,  solution  is  made, 
and  with  it  the  titration  is  practised.  After  the  bouillon 
lias  been  deprived  of  all  coagulable  albumin  and  blood 
coloring  matter  by  boiling  and  filtration,  and  has  cooled 
down  to  the  temperature  of  the  air,  its  whole  volume  is 
exactly  measured. 

From  it  a  sample  of  exactly  5  or  10  c.c.  is  tln-n 
taken,  and  to  it  a  few  drops  of  one  of  the  indicators  com- 
monly employed  in  analytical  work  is  added.  Schnltz 
recommends  1  drop  of  phenolphtalein  solution  (1 
gramme  phenolphtalein  in  300  c.c.  of  alcohol)  to  1  e.e. 
of  bouillon.  The  beaker  containing  the  sample  is 
placed  upon  white  paper,  and  the  dilute  caustic  soda 
solution  is  then  allowed  to  drop  into  it,  very  slowly, 
from  a  burette,  until  there  appears  a  very  delicate  rose 
color,  which  indicates  the  beginning  of  alkaline  reaction. 
A  second  sample  of  the  bouillon  is  treated  in  the  same 
way.  If  the  amounts  of  soda  solution  required  for 
each  sample  deviate  but  very  slightly  or  not  at  all 
the  one  from  the  other,  the  mean  of  these  amounts  is 
taken  as  the  amount  of  the  soda  solution  necessary  to 
neutralize  the  quantity  of  bouillon  employed.  If  10  c.c. 


BOUILLON.  61 

of  bouillon  were  employed,  then,  for  the  whole  amount 
of  1  litre,  just  100  times  as  much,  minus  that  for  the  two 
samples  used  in  titration,  will  be  needed.  For  example  : 
To  neutralize  10  c.c.  of  bouillon,  2  c.c.  of  the  diluted 
(0.4  per  cent.)  caustic  soda  solution  were  employed.  For 
tne  remaining  980  c.c.  of  the  litre  of  bouillon,  then, 
200  c.c.  ( — 4  c.c.,  the  amount  employed  for  the  two 
samples  of  10  c.c.  each  of  bouillon)  is  needed  of  the  0.4 
per  cent,  solution,  or  one-tenth  of  this  amount  of  the  4 
per  cent,  solution. 

For  the  neutralization  of  the  whole  bulk  of  the  bouillon 
it  is  better  to  employ  the  strong  alkaline  solution,  as  by 
its  use  the  volume  is  not  increased  to  so  great  an  extent 
as  when  the  dilute  solution  is  used. 

It  is  evident  that  this  method  is  much  more  exact  than 
that  ordinarily  employed,  but  at  the  same  time  it  must 
be  remembered  that  for  its  success  it  requires  exactness 
in  the  measurement  of  the  volumes  and  the  preparation 
of  the  dilutions.  To  obviate  error,  it  is  better  to  employ 
this  method  when  the  solutions  are  all  cool  and  of  nearly 
the  same  temperature,  so  that  rapid  fluctuations  in  tem- 
perature, and  consequent  alterations  in  volume,  will  not 
materially  interfere  with  the  accuracy  of  the  results. 

This  method  of  neutralization,  which  is  employed  by 
Schultz,  is  to  be  recommended  for  those  experiments  in 
which  slight  inaccuracies  in  the  reaction  of  the  media 
play  an  important  part. 

For  the  ordinary  purposes  of  the  beginner,  however, 
results  quite  satisfactory  in  their  nature  may  be  obtained 
by  the  employment  of  the  saturated  soda  solution  for 
neutralization  and  the  litmus  paper  as  the  indicator. 
For  some  time,  however,  it  has  been  our  practice  to 

4 


62  BACTERIOLOGY. 

employ  the  yellow  curcuma  paper  for  the  detection  of 
alkalinity  rather  than  the  red  litmus  paper. 

Not  infrequently  the  filtered  bouillon,  neutralized  and 
sterilized,  will  be  seen  to  contain  a  fine,  fioceulent  pre- 
cipitate. This  may  be  due  either  to  excess  of  alkalinity 
or  to  incomplete  precipitation  of  the  albumin.  The 
former  may  be  corrected  with  dilute  acetic  or  hydro- 
chloric acid,  and  the  bouillon  again  boiled,  filtered,  and 
sterilized ;  or,  if  due  to  the  latter  cause,  subsequent 
boiling  and  filtration  usually  results  in  ridding  the 
bouillon  of  the  precipitate. 

NUTRIENT  GELATIN.— For  the  preparation  of  gela- 
tin the  bouillon  is  first  prepared  in  exactly  the  same 
way  as  has  just  been  described,  except  that  the  neutral- 
ization takes  place  after  the  gelatin  has  been  completely 
dissolved,  which  occurs  very  rapidly  in  hot  bouillon. 
The  reaction  of  the  gelatin  as  it  comes  from  the  manu- 
factories is  usually  quite  acid,  so  that  a  much  larger 
amount  of  alkali  is  needed  for  its  neutralization  thau  for 
other  media.  The  gelatin  is  added  in  the  proportion  of 
from  10  to  12  per  cent.  The  complete  solution  of  the 
gelatin  may  be  accomplished  either  over  the  water-bath, 
in  the  steam  sterilizer,  or  over  a  free  flame.  If  the  latter 
method  is  practised,  care  must  be  given  that  the  mixture 
is  constantly  stirred  to  prevent  burning  at  the  bottom 
and  consequent  breaking  of  the  flask,  if  a  flask  is  em- 
ployed. 

For  some  time  it  has  been  our  practice  to  use,  for  the 
purpose  of  making  both  gelatin  and  agar,  enamelled  iron 
saucepans  instead  of  glass  flasks ;  by  this  means  the  free 
flame  may  be  employed  without  danger  of  breaking  the 
vessel,  and,  with  a  little  care,  without  fear  of  burning 
the  media.  Under  any  conditions  it  is  better  to  protect 


NUTEIENT    GELATIN.  63 

the  bottom  of  the  vessel  from  the  direct  action  of  the 
flame  by  the  interposition  of  several  layers  of  wire  .gauze 
or  a  thin  sheet  of  asbestos-board. 

When  the  gelatin  is  completely  melted,  it  may  be 
filtered  through  a  folded  paper  filter  on  an  ordinary 
funnel ;  if  the  solution  is  perfect,  this  should  be  very 
quickly  accomplished. 

The  employment  of  the  hot-water  funnel,  so  often 
recommended,  has  been  dispensed  with  in  this  work 
to  a  very  large  extent,  as  we  know  that,  if  the  solution 
of  the  gelatin  is  complete,  filtration  is  so  rapid  as  not 
to  necessitate  the  use  of  an  apparatus  for  maintaining 
the  high  temperature.  The  temperature  at  which  the 
hot-water  funnel  retains  the  gelatin  is  so  high  that  evap- 
oration and  condensation  rapidly  occur,  and  in  conse- 
quence the  filtration  is,  as  a  rule,  retarded.  The  filtra- 
tion is  frequently  done  in  the  steam  sterilizer,  but  this  is 
unnecessary  if  the  gelatin  is  quite  dissolved.  At  the 
ordinary  temperature  of  the  room  and  by  the  means  com- 
monly employed  for  the  filtration  of  other  substances, 
both  gelatin  and  agar-agar  may  be  rapidly  filtered  if 
they  are  completely  dissolved. 

It  not  infrequently  occurs  that,  even  under  the  most 
careful  treatment,  the  filtered  gelatin  is  not  perfectly 
transparent  (the  condition  in  which  it  must  exist,  other- 
wise it  is  useless),  and  clarification  becomes  necessary. 
For  this  purpose  the  mass  must  be  redissolved,  and 
when  at  a  temperature  between  60°  C.  and  70°  C.,  the 
whites  of  two  eggs,  which  have  been  beaten  up  with 
about  50  c.c.  of  water,  are  added.  The  whole  is  then 
thoroughly  mixed  together  and  again  brought  to  the 
boiling-point,  and  kept  at  this  point  until  coagulation  of 
the  albumin  occurs.  It  is  better  not  to  break  up  the 


64  "   BACTERIOLOGY. 

large  masses  of  coagulated  albumin  if  it  can  be  avoided, 
as  when  broken  up  into  fine  flakes  they  clog  the  filter 
and  render  filtration  very  difficult. 

The  practice  sometimes  recommended  of  removing 
these  albuminous  masses  by  first  filtering  the  gelatin 
through  a  cloth,  and  then  finally  through  paper,  is  not 
only  superfluous,  but  in  most  instances  renders  the  pro- 
cess of  filtration  much  more  difficult,  because  of  the 
disintegration  of  these  masses  into  the  finer  particles, 
which  have  the  effect  just  mentioned. 

Under  no  circumstances  is  a  filter  to  be  used  for  these 
purposes  without  first  having  been  moistened  with  water. 
If  this  is  not  done,  the  pores  of  the  paper,  which  are 
relatively  large  when  in  a  dry  state,  when  moistened  by 
the  gelatin  not  only  diminish  in  size,  but  in  contract- 
ing are  often  entirely  occluded  by  the  finer  albumin- 
ous flakes  which  become  fixed  within  them.  In  this 
way  the  filter  may  become  almost  entirely  occluded. 
The  preliminary  moistening  with  water  causes  the 
diminution  of  the  size  of  the  pores  to  such  an  extent 
that  the  finer  particles  of  the  precipitate  now  rest  on  the 
surface  of  the  paper,  instead  of  becoming  fixed  in  its 
meshes. 

During  boiling  it  is  well  to  filter  from  time  to  time 
a  few  cubic  centimetres  of  the  gelatin  into  a  test- tube 
and  boil  it  over  a  free  flame  for  a  minute  or  so ;  in 
this  way  one  can  detect  if  all  the  albumin  has  been 
coagulated  and  when  the  solution  is  ready  for  filtration. 

Gelatin  should  not,  as  a  rule,  be  boiled  over  ten  or 
fifteen  minutes  at  one  time,  or  left  in  the  steam  sterilizer 
for  more  than  thirty  to  forty-five  minutes,  otherwise  its 
property  of  solidifying  is  materially  diminished. 

As  soon  as  the   gelatin  is  complete,  whether  it  is 


NUTRIENT    AGAR-AGAR.  65 

retained  in  the  flask  into  which  it  has  been  filtered  or 
decanted  off  into  sterilized  test-tubes,  it  should  be  steril- 
ized in  the  steam  sterilizer  on  three  successive  days,  for 
fifteen  minutes  each  day — the  mouth  of  the  flask  or  the 
test-tubes  containing  it  having  been  previously  closed 
with  cotton  plugs. 

NUTRIENT  AGAR-AGAR. — The  preparation  of  the 
nutrient  agar-agar  by  the  beginner  is,  as  a  rule,  a  some- 
what tedious  and  time-taking  experience.  This  is  owing 
mainly  to  lack  of  patience  and  failure  to  adhere  strictly 
to  the  rules  laid  down  for  the  preparation  of  this  medium. 
Many  methods  are  recommended  for  its  preparation  ; 
almost  every  worker  has  some  slight  modification  of  his 
own. 

The  method  which  has  given  the  best  results  in  our 
hands,  and  from  which  there  are  no  grounds  for  devi- 
ating, is  as  follows  : 

Prepare  the  bouillon  in  the  usual  way.  Agar-agar 
reacts  neutral,  so  that  the  bouillon  may  be  neutralized 
before  the  agar  is  added.  Then  add  finely-chopped 
agar  in  the  proportion  of  1  to  1.5  per  cent.  Place 
the  mixture  in  a  porcelain-lined  iron  vessel  and  make  a 
mark  on  the  sides  of  the  vessel  at  which  the  level  of  the 
fluid  stands,  add  about  250  c.c.  of  water  and  allow  the 
mass  to  boil  slowly,  occasionally  stirring,  over  a  free 
flame  for  three  or  four  hours.  Care  must  be  given  that 
it  does  not  boil  over  the  sides  of  the  vessel.  From  time 
to  time  observe  if  the  fluid  has  fallen  below  the  mark  of 
its  original  level ;  if  it  has,  add  water  until  its  original 
volume  is  restored.  At  the  end  of  the  time  given  re- 
move the  flame  and  place  the  vessel  containing  the  mix- 
ture in  a  large  dish  of  cold  water ;  stir  the  agar  con- 
tinously  until  it  has  cooled  down  to  about  68°-70°  C., 


66  BACTERIOLOGY. 

and  then  add  the  whites  of  two  eggs  which  have  been 
beaten  up  in  about  50  c.c.  of  water.  Mix  this  carefully 
throughout  the  agar,  and  allow  the  mass  to  boil  slowly 
for  about  one-half  hour,  observing  all  the  while  the  level 
of  the  fluid.  It  is  necessary  to  reduce  the  temperature 
of  the  mass  to  the  extent  given,  68°-70°  C.,  otherwise 
the  coagulation  of  the  albumin  will  occur  in  lumps  and 
masses  as  soon  as  it  is  added,  and  its  clearing  action  will 
not  be  homogeneous.  The  process  is  a  purely  mechani- 
cal one — the  finer  particles,  which  would  otherwise  pass 
through  the  pores  of  the  filter,  being  taken  up  by  the 
albumin  as  it  coagulates  and  retained  in  the  coagula. 

At  the  end  of  one- half  hour  the  boiling  mass  may  be 
easily  and  quickly  filtered  through  a  heavy,  folded  paper 
filter  at  the  room  temperature,  and,  as  a  rule,  the  filtrate 
is  as  clear  and  as  transparent  as  agar-agar  usually  appears. 
If  the  mixture  is  positively  alkaline,  it  is  not  only  cloudy, 
but  it  filters  with  difficulty  ;  if  it  is  acid,  it  is  usually 
quite  clear ;  but,  as  Schultz  has  pointed  out,  it  loses  at 
the  same  time  some  of  its  gelatinizing  properties.  The 
bouillon  should  always  be  neutralized  before  the  agar- 
agar  is  added  to  it,  for  the  bouillon,  which  is  normally 
acid,  from  the  acid  of  the  meat,  robs  the  agar,  under 
the  influence  of  heat,  of  some  of  its  gelatinizing  powers  ; 
this  cannot  be  regained  by  subsequent  neutralization. 

Another  method  by  which  the  agar-agar  can  easily 
and  quickly  be  melted,  is  by  steam  under  pressure.  If 
the  flask  containing  the  mixture  of  bouillon  and  agar 
be  kept  in  the  digestor  or  autoclav,  with  the  steam  under 
a  pressure  of  one  atmosphere,  as  shown  by  the  gauge,  for 
from  twenty  to  thirty  minutes,  the  agar-agar  will  be 
found  at  the  end  of  this  time  completely  melted,  and  fil- 
tration may  then  be  accomplished  with  but  little  difficulty. 


PREPARATION  OF  POTATOES.        67 

If  glycerin  is  to  be  added  to  the  agar-agar,  it  is  done 
after  filtration  and  before  sterilization.  The  nutritive 
properties  of  the  media  for  certain  organisms,  particu- 
larly the  tubercle  bacillus,  is  improved  by  the  addition 
of  glycerin  in  the  proportion  of  5  to  7  per  cent. 

If  after  filtration  a  fine  flocculent  precipitate  is  seen, 
look  to  the  reaction  of  the  medium.  If  it  is  quite  alka- 
line, neutralize,  boil,  and  filter  again.  If  the  reaction 
is  neutral  or  only  very  slightly  acid,  dissolve  and  clarify 
again  with  egg  albumin  by  the  method  given. 

The  most  important  point  in  all  the  media,  aside 
from  the  correct  proportion  of  the  ingredients,  is  their 
reaction.  They  must  be  neutral  or  very  slightly  alka- 
line. But  few  organisms  develop  well  on  media  of  an 
acid  reaction.  In  all  of  the  above  media  the  meat  extracts 
now  on  the  market  may  occasionally  be  substituted  for 
the  meat  itself  in  preparing  the  bouillon.  In  this  case 
the  preparation  known  as  Liebig's  Meat  Extract  may  be 
employed  in  the  proportion  of  from  three  to  five  grammes 
to  the  litre  of  water. 

PREPARATION  OF  POTATOES. — Potatoes  are  prepared 
for  use  in  two  ways : 

1.  They  are  taken  as  they  come  to  the  market — old 
potatoes  being  usually  recommended,  and  carefully 
scrubbed  under  the  water-tap  with  a  stiff  brush  until 
all  adherent  dirt  has  been  removed  ;  "  the  eyes  "  and  all 
discolored  or  decayed  parts  are  carefully  removed  with 
a  pointed  knife.  They  are  then  to  be  placed  in  a  solu- 
tion of  corrosive  sublimate  of  the  strength  of  1:1000 
and  allowed  to  remain  there  for  twenty  minutes ;  at  the 
end  of.this  time,  without  rinsing  off  the  sublimate,  they 
are  placed  into  a  covered  tin  bucket  with  a  perforated 
bottom  and  sterilized  in  the  steam  sterilizer  for  forty- 


68  BACTERIOLOGY. 

five  minutes.  On  the  second  and  third  days  the  sterili- 
zation is  repeated  for  fifteen  to  twenty  minutes  each 
day.  They  must  not  be  removed  from  the  sterilizing 
bucket  until  sterilization  is  complete.  At  the  end  of 
this  time  they  are  ready  for  use.  When  prepared  in 
this  way,  they  are  usually  intended  to  be  cut  into  two 
halves,  and  the  cultivation  of  the  organisms  is  to  be 
conducted  upon  the  flat  surfaces  of  the  sections. 

This  method  requires  some  care  to  prevent  con- 
tamination during  manipulation.  The  hand  which  is 
to  take  up  the  potato  from  the  bucket,  which  until 
now  has  remained  covered,  is  first  disinfected  in  the 
sublimate  solution  for  ten  minutes,  the  potato  is  then 
taken  up  between  the  thumb  and  index  finger,  and  sev- 
ered into  two  by  a  knife  which  has  just  been  sterilized 
in  the  free  flame  until  it  is  quite  hot.  The  blade  of  the 
knife  is  passed  not  quite  through  the  potato,  but  nearly 
so.  A  large  glass  culture-dish  for  the  reception  of  the 
two  halves  of  the  potato  having  been  disinfected  for 
twenty  minutes  with  1  :  1000  sublimate  solution  and 
then  drained  of  all  the  adherent  solution,  is  at  hand 
ready  for  the  bits  of  potato ;  the  cover  is  removed,  and 
by  twisting  the  knife  gently  the  two  halves  of  the  potato 
may  be  caused  to  fall  apart  in  the  dish  and  usually  to  fall 
upon  their  convex  surfaces,  leaving  the  flat  sections  up- 
permost. The  cover  is  placed  upon  the  dish  and  the 
potatoes  are  ready  for  inoculation. 

2.  Preparation  of  potatoes  jor  test-tube  cultures.  If 
the  potatoes  are  to  be  employed  for  test-tube  cultures, 
one  simply  scrubs  off  the  coarser  particles  of  dirt 
with  water  and  a  brush,  and  with  a  cork-borer  punches 
out  cylindrical  bits  of  potato  which  will  fit  loosely  into 
the  test-tubes  to  He  used.  On  each  bit  of  potato  is  then 


FIG.  6. 


BLOOD-SERUM.  69 

to  be  cut  a  slanting  surface  running  diagonally  from  about 
the  junction  of  the  first  and  second  third  of  the  cylinder 
to  the  diagonally  opposite  end.  These  cylinders  of  potato 
are  now  to  be  left  in  running  water  over  night,  otherwise 
they  are  very  much  discolored  by  the  sterilization  to 
which  they  are  to  be  subjected.  At  the  end  of  this  time 
they  are  placed  into  previously  prepared  test-tubes,  one 
piece  in  each  tube,  with  the  slanting  surface  up,  the  cot- 
ton plugs  of  the  tubes  replaced  and  they  are  then  to  be 
sterilized  in  the  steam  for  forty-five  minutes.  On  the 
second  or  third  day  they  are  to  be  sterilized 
for  fifteen  to  twenty  minutes  each  day. 

The  entire  sterilization  may  be  accom- 
plished in  the  autoclav  with  the  steam  under 
a  pressure  of  one  atmosphere,  by  a  single 
exposure  of  twenty  to  twenty-five  minutes. 
When  finished  they  have  the  appearance  seen 
in  Fig.  6. 

Care  must  be  given  to  the  steriliza- 
tion of  potatoes,  because  they  always  have 
adhering  to  them  the  organisms  commonly 
found  in  the  ground,  the  spores  of  which  are 
among  the  most  resistant  of  all  known  or- 
ganisms. The  so-called  "  potato  bacillus  "  is 
one  of  this  group  ;  it  is  an  organism  which  not 
infrequently  is  more  or  less  of  an  obstacle 
to  the  work  of  the  beginner. 

BLOOD-SERUM.  —  Blood  -  serum    requires 
special  care  in  its  preparation ;   it  is  desir- 
able under   all   conditions  to   reduce  the  unavoidable 
contamination  which  to  a  certain  extent  occurs  during 
the  manipulation,  to  its  minimum  degree. 

It  is  possible  to  collect  serum  from  small  animals  and 
4* 


70  BACTERIOLOGY. 

in  small  quantities  under  such  precautions  that  it  is 
perhaps  not  contaminated,  but  ordinarily  for  laboratory 
purposes  a  larger  quantity  is  needed,  so  that  the  slaughter- 
houses form  the  sources  from  which  it  is  usually  ob- 
tained, and  here  a  certain  amount  of  contamination  is 
unavoidable,  though  its  degree  may  be  limited  by  proper 
precaution.  The  animal  from  which  the  blood  is  to  be 
collected  should  be  drawn  up  to  the  ceiling  by  the  hind 
legs,  the  head  should  be  held  well  back,  and  with  one 
pass  of  a  very  sharp  knife  the  throat  should  be  com- 
pletely cut  through.  The  blood  which  will  be  spurting 
from  the  severed  vessels  should  be  collected  in  large 
glass  jars  which  have  been  previously  cleaned,  disinfected^ 
and  all  traces  of  the  disinfectant  removed  with  alcohol 
and  finally  ether.  The  latter  evaporates  very  quickly 
and  leaves  the  jar  quite  dry.  The  jars  should  be  pro- 
vided with  covers  which  close  hermetically — these  too 
should  be  carefully  disiufected.  The  best  form  of  glass 
vessels  for  the  purpose  are  the  large  glass  museum  jars 
of  about  one  gallon  capacity,  which  close  by  a  cover 
which  can  be  tightly  screwed  down  upon  a  rubber  joint. 
From  two  such  jarfuls  of  blood  one  can  recover  quite 
a  large  quantity  of  serum,  ordinarily  from  500-700  c.c. 
The  jars  having  been  filled  with  blood,  their  covers  are 
placed  loosely  upon  them  and  they  are  allowed  to  stand 
for  about  fifteen  minutes  until  clotting  has  begun.  At 
the  end  of  this  time  a  clean  glass  rod  is  passed  around 
the  edges  of  the  surface  of  the  clot  to  break  up  any 
adhesions  to  the  wall  of  the  jar  that  might  have  formed, 
and  which  would  prevent  the  sinking  of  the  clot  to  the 
bottom.  The  covers  are  then  tightly  replaced,  and  witl i  a- 
little  agitation  as  possible  the  jars  are  placed  in  an  ice- 
chest,  where  they  remain  for  twenty-four  to  forty-eight 


BLOOD-SERUM.  71 

hours.  The  temperature  should,  however,  not  be  low 
enough  to  prevent  coagulation,  but  should  be  sufficiently 
low  to  interfere  with  the  development  of  any  living  or- 
ganisms that  may  be  present.  The  temperature  of  the 
ordinary  domestic  refrigerator  is  sufficient  for  the  purpose. 
After  twenty-four  to  forty- eight  hours  the  clot  will  have 
become  firm,  and  will  be  seen  at  the  bottom  of  the  jar. 
Above  it  is  a  quantity  of  dark  straw-colored  serum. 
The  serum  may  then  be  drawn  off  with  a  sterilized 
pipette  and  placed  in  tall  cylinders  which  have  previ- 
ously been  plugged  with  cotton  wadding  and  sterilized. 
After  treating  all  the  serum  in  this  way,  care  having 
been  taken  to  get  as  little  of  the  coloring  matter  of  the 
blood  as  possible,  it  may  be  placed  again  in  the 
ice-chest  for  twenty- four  hours  during  which  time  the 
corpuscular  elements  will  sink  to  the  bottom,  leaving 
the  supernatant  fluid  quite  clear.  This  may  then  be 
pipetted  off,  either  into  sterilized  test-tubes,  about  8  c.c. 
to  each  tube,  or  into  small  sterilized  flasks  of  about  100 
c.c.  capacity.  It  is  then  to  be  sterilized  by  the  inter- 
mittent method  at  low  temperatures,  viz.,  for  one  hour  on 
each  of  five  consecutive  days  at  a  temperature  of  68°-70° 
C.  During  the  intervening  days  it  is  to  be  kept  at  the 
room  temperature  to  permit  of  the  development  of  any 
spores  that  may  be  present  into  their  vegetative  forms, 
in  which  condition  they  are  killed  by  an  hour's  exposure 
to  the  temperature  of  70°  C. 

At  the  end  of  this  time  the  serum  in  the  tubes  may 
either  be  retained  as  fluid  serum  or  solidified  at  between 
76°-80°  C.  In  solidifying  the  serum  the  tubes  should 
be  placed  in  an  inclined  position  so  that  as  great 
a  surface  as  possible  may  be  given  to  the  serum.  The 
process  of  solidification  requires  constant  attention  if 


72  BACTERIOLOGY. 

good  results  are  to  be  obtained,  i.  e.,  if  a  translucent, 
solid  medium  is  to  result.  If  the  old,  small  form  of 
apparatus  is  employed  (Fig.  7),  then  the  solidification 
can  be  accomplished  in  a  shorter  time  than  if  the  larger 
forms,  which  are  now  frequently  employed,  are  used. 
No  definite  rule  for  the  time  that  will  be  required  can 
be  laid  down,  for  this  is  not  constant.  If  the  small 
solidifying  apparatus  is  used,  very  good  results  may  be 
obtained  in  about  two  hours  at  78°  C.  It  frequently 
requires  a  longer  time  at  a  higher  temperature  than 
has  been  mentioned  This  is  especially  the  case  with 
Loffler's  serum  mixture. 

FIG.  7. 


The  best  results  are  obtained  when  a  low  temperature 
is  employed  for  a  long  time.  Under  any  circumstances 
the  tubes  must  be  observed  from  time  to  time  through 
the  glass  door  or  cover  with  which  the  solidifying  oven 
is  provided,  and  each  time  the  oven  should  be  slightly 


BLOOD-SERUM.  73 

jarred  with  the  hand  to  see  if  solidification,  as  indi- 
cated by  the  disappearance  of  tremors  from  the  serum,  is 
beginning.  If  the  temperature  gets  too  high,  or  the  ex- 
posure is  too  long,  an  opaque  medium  results.  The  tem- 
perature to  be  observed  is  that  of  the  air  inside  the  cham- 
ber, and  also  that  of  the  water  surrounding  it.  The  latter 
is  usually  a  degree  or  two  higher  than  the  former.  The 
tubes  should  not  rest  directly  upon  the  heated  bottom  or 
against  the  heated  sides  of  the  chamber,  but  should  lie 
upon  racks  of  wood  or  wire,  and  be  protected  from  the 
sides  by  a  wire  screen  of  gauze ;  in  this  way  the  tubes 
are  all  exposed  to  about  the  same  temperature.  The 
thermometer  which  indicates  the  temperature  inside  the 
chamber  should  not  touch  the  surfaces  but  should  either 
be  suspended  free  from  above  through  a  cork  in  the  top 
of  the  apparatus,  if  the  large  form  of  apparatus  is  used, 
or  should  lie  upon  a  rack  of  cork  or  wood,  its  bulb  being 
free  and  a  little  lower  than  the  other  extremity,  if  the 
small,  old-fashioned  apparatus  of  Koch  is  employed. 
The  latter  form  is  preferable,  as  it  is  more  easily  man- 
aged. 

When  solidification  is  complete,  the  tubes  are  to  be 
retained  in  the  erect  position  and,  unless  they  are 
intended  for  immediate  use,  must  be  prevented  from  dry- 
ing. The  superfluous  ends  of  the  cotton  plugs  should  be 
^  burned  oif,  and  the  mouths  of  the  tubes  should  then  be 
covered  by  sterilized  rubber  caps.  Even  with  the  greatest 
care,  it  not  uncommonly  happens  that  one  or  two  of  the 
lot  of  tubes  thus  prepared  and  protected  will  become  con- 
taminated. This  is  usually  due  to  spores  of  moulds  that 
have  fallen  into  the  rubber  caps  or  on  the  cotton  plugs 
during  manipulation,  and,  finding  no  means  of  outward 
growth,  have  thrown  their  hyphen  downward  through 


74  BACTERIOLOGY. 

the  cotton  into  the  tube,  and  their  spores  have  fallen  upon 
the  surface  of  the  serum  and  gone  on  to  develop. 

SPECIAL  MEDIA. — The  media  just  described — bou- 
illon, nutrient  gelatin,  nutrient  agar-agar,  potato,  ;m<l 
blood-serum — are  those  in  general  use  in  the  laboratory 
for  purposes  of  isolation  and  study  of  the  ordinary  forms 
of  bacteria.  For  the  finer  points  of  differentiation 
special  media  have  been  suggested;  a  few  of  them  will 
be  mentioned. 

Milk.  Fresh  milk  should  be  allowed  to  stand  over 
night  in  the  ice-chest,  the  cream  then  removed,  and  the 
remainder  of  the  milk  pipetted  into  test-tubes,  about 
8  c.c.  to  each  tube,  and  sterilized  by  the  intermittent 
process,  at  the  temperature  of  steam,  for  three  successive 
days. 

The  cream  is  best  separated  from  the  milk  by  the  use 
of  a  cylindrical  vessel  with  stop-cock  at  the  bottom,  by 
means  of  which  the  milk,  devoid  of  cream,  may  be  drawn 
off.  A  Chevallier  creamometer  with  stop-cock  at  the 
bottom  serves  the  purpose  very  well.  It  should  be 
covered  while  standing. 

Milk  may  be  used  as  a  culture  medium  without 
any  addition  to  it,  or,  before  sterilizing,  a  few  drops  of 
litmus  tincture  may  be  added,  just  enougir  to  give  it  a 
pale  blue  color.  By  this  means  it  may  be  seen  that  dif- 
ferent organisms  bring  about  different  reactions  in  the 
medium ;  some  producing  alkalies  which  cause  the  blue 
color  to  be  intensified,  others  producing  acids  which 
change  it  to  red,  while  others  bring  about  neither  of 
these  changes. 

Milk  may  also  be  employed  as  a  solid  culture  medium 
by  the  addition  to  it  of  gelatin  or  agar-agar  in  the  pro- 
portions given  for  the  preparation  of  the  ordinary  uutri- 


SPECIAL    MEDIA.  75 

ent  gelatin  or  agar-agar.  It  has,  however,  in  this  form 
the  disadvantage  of  not  being  transparent,  and  can  there- 
fore best  be  used  for  the  study  of  those  organisms  which 
grow  upon  the  surface  of  the  medium  without  causing 
liquefaction. 

Nutrient  gelatin  and  agar-agar  can  also  be  prepared 
from  neutral  milk  whey,  obtained  from  milk  by  pre- 
cipitation of  the  casein. 

Dunham's  solution  and  peptone-rosalic-acid  solution. 
Peptone  solution,  to  which  rosalic  acid  has  been  added, 
also  serves  very  well  for  the  detection  of  alterations  in 
reaction.  The  peptone  solution  of  Dunham  is  the  form 
that  we  have  usually  employed.  It  consists  of 

Distilled  water   ......     100  parts. 

Dried  peptone 1  part. 

Sodium  chloride         .        .         .         .         .        0.5    " 

and  4  c.c.  of  the  following  solution  : 

Rosalie  acid  (coralline)       ....          0.5  gramme. 
Alcohol  (80  per  cent.)        .         .         .         .100  c.c. 

This  is  to  be  boiled,  filtered,  and  decanted  into  clean, 
sterilized  test-tubes,  about  8  to  10  c.c.  to  each  tube.  The 
tubes  are  then  to  be  sterilized  in  the  usual  way  by  steam. 
When  sterilization  is  completed  and  the  tubes  cooled,  the 
solution  will  be  of  a  very  pale  rose  color,  which  disap- 
pears entirely  under  the  action  of  acids,  and  becomes 
much  more  intense  when  alkalies  are  produced.  We  have 
used  this  solution  for  some  time  for  flie  study  of  the 
reactions  produced  by  different  organisms,  and  find  it 
a  valuable  addition  to  our  means  of  differentiation  of 
bacteria. 

Loffler's  blood-serum  mixture.  Loffler's  blood-serum 
mixture  consists  of  one  part  of  neutral  meat-infusion 


76  BACTERIOLOGY. 

bouillon  containing  1  per  cent,  of  grape-sugar,  and  tl im- 
parts of  blood-serum.  This  mixture  is  placed  in  test- 
tubes,  sterilized,  and  solidified  in  exactly  the  way  given 
for  blood  serum. 

Giiarniari's  cigar-gelatin  : 

Meat-infusion    ......  950  c.c. 

Sodium  chloride         .....         5  grammes. 

Peptone 25-30    " 

Gelatin .  40-60    " 

Agar-agar 3-4       " 

Water       .......  50  c.c. 

The  point  in  the  preparation  of  this  medium  is  its 
reaction,  which  should  be  exactly  neutral. 

The  list  of  special  media  is  too  great  to  be  given  in  a 
work  of  this  size.  Their  description  must  be  seen  in  the 
original.  Those  which  have  been  given  above  will 
suffice  for  obtaining  a  clear  understanding  of  the  prin- 
ciples of  the  work. 

NOTE. — The  term  "meat-infusion"  always  implies  a 
watery  extract  of  meat  made  by  mixing  500  grammes  of 
finely-chopped  lean  meat  and  1  litre  of  water  together, 
and  allowing  them  to  stand  in  a  cool  place  for  twenty- 
four  hours.  At  the  end  of  this  time  the  fluid  portion  is 
strained  off  through  a  coarse  towel.  This  represents  the 
infusion. 


CHAPTER   VII. 

Preparation  of  the  tubes,  flasks,  etc.,  in  which  the  media  are  to  be 

preserved. 

\ 

WHILE  the  media  are  in  course  of  preparation  it  is 
well  to  get  the  test-tubes  and  flasks  ready  for  their  recep- 
tion. It  is  essential  that  the  tubes  for  this  purpose  should 
be  as  clean  as  it  is  possible  to  make  them.  For  this 
purpose  it  is  advisable  that  both  new  tubes  and  those 
which  have  previously  been  used  should  be  boiled  for 
some  time,  about  thirty  to  forty-five  minutes,  in  a  strong  / 
solution  of  common  soda,  about  a  4  or  6  per  cent,  solu- 
tion ;  it  is  not  necessary  to  be  exact  as  to  the  strength, 
but  it  should  not  be  weaker  than  this.  At  the  end  of  this 
time  they  are  to  be  carefully  swabbed  out  with  a  cylin- 
drical bristle  brush,  preferably  one  having  a  reed  handle 
(Fig.  8),  as  those  with  wire  handles  are  apt  to  break 

FIG.  8. 


through  the  bottoms  of  the  tubes.  All  trace  of  adherent 
material  should  be  carefully  removed.  When  the  tubes 
are  quite  clean  they  may  be  rinsed  in  a  warm  solution 
of  commercial  hydrochloric  acid  of  the  strength  of  about 
1  per  cent.  This  is  to  remove  the  alkali.  They  are 
then  to  be  thoroughly  rinsed  in  clear,  running  water,  and 
stood  top  down  until  the  water  has  drained  from  them. 
When  dry  they  are  to  be  plugged  with  raw  cotton.  The 


78  BACTERIOLOGY. 

plugging  with  the  cotton  requires  a  little  practice  before  it 
can  be  properly  done.  The  cotton  should  be  introduced 
into  the  mouths  of  the  tubes  in  such  a  way  that  no  cracks 
or  creases  exist,  but  should  fill  them  quite  regularly  all 
around.  The  plugs  should  be  neither  too  tight. nor  too 
loose,  the  regular  rule  being  that  when  in  position  the 
plug  should  fit  tight  enough  to  just  sustain  the  weight 
of  the  tube  into  which  it  is  placed  when  held  up 
by  the  portion  which  projects  from  and  overhangs  the 
mouth  of  the  tube.  The  tubes  thus  plugged  with  cotton 
are  now  to  be  placed  upright  in  a  wire  basket  and  heated 
for  one  hour  in  the  hot-air  sterilizer  at  a  temperature  of 
about  150°  C.  A  very  good  rule  for  this  process  of 
sterilizing  is  to  observe  the  tubes  from  time  to  time,  and 
as  soon  as  the  cotton  has  become  a  very  light  brown 
color,  not  deeper  than  a  dark-cream  tint,  to  consider 
sterilization  complete.  The  tubes  are  then  removed  and 
allowed  to  cool  down. 

The  cotton  used  for  this  purpose  should  be  the 
ordinary  cotton  batting  of  the  shops,  and  not  absorbent 
cotton,  the  latter  becomes  too  tightly  packed,  and  is, 
moreover,  much  too  expensive  for  this  purpose. 

Care  should  be  taken  not  to  burn  the  cotton,  other- 
wise the  tubes  will  become  coated  with  a  dark-colored 
oily  deposit  which  renders  them  unfit  for  use,  and  they 
will  have  to  be  cleaned  again. 

FILLING  THE  TUBES. — When  the  tubes  are  cold 
they  may  be  filled.  This  is  best  accomplished  by  the 
use  of  a  spherical  form  of  funnel,  such  as  is  shown  in 
Fig.  I).  The  liquefied  medium  is  poured  into  this  funnel, 
which  has  been  carefully  washed,  and  by  pressing  the 
pinch-cock  with  which  the  funnel  is  provided,  the 


FILLING    THE    TUBES.  79 

desired  amount  of  material  (5-10  c.c.)  may  be  allowed 
to  flow  into  the  tubes  held  under  its  opening. 


It  is  not  necessary  to  sterilize  the  funnel,  for  the 
medium  is  to  be  subjected  to  this  process  as  soon  as  it  is 
in  the  test-tubes. 

Care  should  be  given  that  none  of  the  medium  is 
dropped  upon  the  mouth  of  the  test-tube,  otherwise  the 
cotton  plugs  become  adherent  to  the  tube  and  are  not 
only  difficult  to  remove,  but  present  a  very  untidy  ap- 
pearance, and  interfere,  indeed,  with  the  proper  manipu- 
lations. 

As  soon  as  the  tubes  have  been  filled  they  are  to  be 
sterilized  in  the  steam  sterilizer  for  fifteen  minutes  on 


80  BACTERIOLOGY. 

each  of  three  successive  days.  During  the  intervening 
days  they  may  be  kept  at  the  ordinary  room  temperature. 

When  sterilization  is  complete,  and  the  medium  in 
the  tubes  is  still  liquid,  some  of  them  may  be  placed  in  a 
slanting  position,  at  an  angle  of  about  ten  degrees  with 
the  surface  on  which  they  rest,  and  the  medium  allowed 
to  solidify  in  this  position.  These  are  for  the  so-called 
slant  cultures.  The  balance  may  solidify  in  the  erect 
position  ;  these  serve  for  the  plate  cultures. 

For  Esmarch  tubes  not  more  than  5  c.c.  of  material 
should  be  placed  in  each  tube,  as  more  than  this  renders 
the  rolling  difficult  and  irregular. 


CHAPTER    VIII. 

Technique  of  making  plates — Esmarch  tubes,  Petri  plates,  etc. 

PLATES. — The  plate  method  can  be  practised  with 
both  agar-agar  and  gelatin.  It  cannot  be  practised 
with  blood-serum,  because  the  serum,  when  once  solidi- 
fied, cannot  be  again  liquefied. 

Plates  are  usually  referred  to  as  "  a  set."  This  term 
implies  three  individual  plates  each  representing  the 
mixture  of  organisms  in  a  higher  stage  of  dilution.  The 
first  plate  is  known  usually  as  "the  original/'  or  "plate 
1,"  the  first  dilution  from  this  as  "  plate  2,"  and  the 
second  as  "  plate  3." 

In  the  preparation  of  a  set  of  plates  the  following  are 
the  steps  to  be  observed  : 

Three  tubes,  each  containing  from  7  to  9  c.c.  of  gela- 
tin or  agar-agar,  are  placed  in  the  water-bath  until  the 
medium  has  become  liquid.  If  agar-agar  is  employed, 
this  is  accomplished  at  the  boiling-point  of  water;  if 
gelatin  is  used,  a  much  lower  temperature  suffices  (35°- 
40°  C.).  When  liquefaction  is  complete  the  temperature 
of  the  water,  in  the  case  of  agar-agar,  must  be  reduced 
to  41°— 42°  C.,  at  which  temperature  the  agar-agar  re- 
mains liquid  and  the  organisms  may  be  introduced  into 
it  without  fear  of  destroying  their  vitality.  The  medium 
being  now  liquid  and  of  the  proper  temperature,  a  very 
small  portion  of  the  mixture  of  organisms  to  be  studied 
is  taken  up  with  a  sterilized,  looped  platinum  wire,  an 


82  BACTERIOLOGY. 

"oese"  as  it  is  called,  Fig.  10.  This  is  nothing  more  than 
a  piece  of  platinum  wire  of  about  5  c.m.  long,  twisted  into 
a  small  loop  at  one  end  and  fused  into  a  bit  of  glass  rod, 
which  acts  as  a  handle,  at  the  other  extremity.  This 
aoese"  is  one  of  the  most  useful  of  bacteriological  instru- 
ments, as  there  is  hardly  a  manipulation  in  the  work  into 
which  it  does  not  enter.  Under  no  conditions  is  it  to  be 

FIG.  10. 


employed  without  having  been  passed  through  the  gas- 
flame  until  quite  hot ;  this  is  for  the  purpose  of  sterili- 
zation. One  should  form  a  habit  of  never  taking  up 
one  of  these  "oeses,"  or  platinum- wire  needles,  as  they 
are  also  called,  for  they  are  both  looped  and  curved  or 
straight,  without  passing  it  through  the  flame,  and  the 
sooner  the  beginner  learns  to  do  this  as  a  matter  of 
reflex,  the  sooner  does  he  rid  himself  of  one  of  the  pos- 
sible sources  of  error  in  his  work.  It  must  be  remem- 
bered, though,  that  the  "  oese  "  should  not  be  used  when 
hot,  otherwise  the  organisms  taken  up  with  it  are  killed 
by  the  high  temperature;  after  sterilizing  it  in  the  flame 
one  waits  for  a  few  seconds  before  using  it. 

The  bit  of  material  under  consideration  is  transferred 
with  the  sterilized  "  oese "  into  tube  No.  1,  "the 
original,"  where  it  is  carefully  disintegrated  by  gently 
rubbing  it  against  the  sides  of  the  tube.  The  more 
carefully  this  is  done  the  more  homogeneous  will  be  the 


TECENIQUE  OF  MAKING  PLATES.     83 

distribution  of  the  organisms  and  the  better  the  results. 
The  "oese"  is  then  again  sterilized,  and  three  of  its 
loopfuls  are  passed,  without  touching  the  sides  of  the 
tube,  from  "  the  original  "  into  tube  No.  2,  where  they 
are  carefully  mixed.  Again  the  "oese"  is  sterilized 
and  again  three  dips  are  made  from  tube  2  into  tube  3. 
This  completes  the  dilution.  The  " 
ized  before  laying  it  aside. 

FIG.  11. 


During  this  manipulation,  which  must  be  done  quickly 
if  agar-agar  is  employed,  the  temperature  of  the  water  in 
the  bath  in  which  the  tubes  stand  should  never  get  lower 
than  39°  C.,  and  never  higher  than  43°  C.  If  it  falls 
too  low,  below  38°  C.,  the  agar  gelatinizes,  and  can 
only  be  redissolved  by  a  temperature  which  would  be 
destructive  to  the  organisms  which  may  have  been  in- 
troduced into  the  tubes.  This  is  not  of  so  much 
moment  with  gelatin,  as  it  may  readily  be  redissolved 


84  BACTERIOLOGY. 

at  a  temperature  not  detrimental  to  the  organisms  with 
which  the  tubes  may  have  been  inoculated 

THE  COOLING-STAGE  AND  LEVELLING  TRIPOD. — 
While  the  medium  of  which  the  plates  are  to  be  made  is 
melting,  it  is  well  to  arrange  the  cooling-stage  (Fig.  11) 
upon  which  it  is  to  be  subsequently  solidified. 

This  stage  consists  of  a  glass  dish  filled  with  ice-water 
and  covered  with  a  ground-glass  plate,  which  in  turn  has 
a  dome-shaped  cover.  The  dish  rests  upon  a  tripod 
which  can  be  brought  to  an  exact  level,  as  indicated  by 
the  spirit-level,  by  raising  or  lowering  its  legs  by  means 
of  screws,  with  which  they  are  provided.  Three  stages 
are  usually  employed.  When  ready  for  use  they  should 
be  exactly  level. 

THE  GLASS  PLATES. — On  the  stages  are  to  be  placed 
the  glass  plates  upon  which  the  liquefied  gelatin  or  agar- 
agar  is  to  be  poured  and  allowed  to  solidify.  It  is 

FIG.  12. 


therefore  necessary  that  the  plates  should  not  only  be 
sterile  when  placed  upon  the  stages,  but  should  l>e 
carefully  protected  by  a  cover  against  dust  aud  bacteria 
from  outside  sources  during  manipulation. 

A  number  of  plates  at  a  time  are  usually  sterilized  in 
the  dry  sterilizer  at  a  temperature  of  150°  to  180°  C. 
for  one  hour.  During  sterilization  and  until  used  they 


TECHNIQUE    OF    MAKING    PLATES.  85 

are  retained  in  an  iron  box  (Fig.  12),  which  is  especially 
designed  for  the  purpose. 

They  should  never  be  placed  upon  the  stage  until 
cold,  otherwise  they  crack. 

When  the  plates  which  have  been  placed  upon  the 
stages  are  quite  cold,  the  melted  gelatin  or  agar-agar  in 
the  tubes  which  represent  the  three  dilutions  should  be 
poured  upon  them,  each  tube  being  emptied  upon  a 
separate  plate.  If  the  medium  is  quite  fluid  it  spreads 
over  the  surface  of  the  plates  in  a  thin  even  layer. 
Sometimes  it  may  be  more  evenly  spread  as  it  flows 
from  the  tube  by  the  aid  of  a  sterilized  glass  rod. 

As  the  contents  of  each  tube  is  emptied  upon  a  plate 
the  cover  of  the  cooling-stage  is  quickly  replaced  and 
the  plate  allowed  to  stand  until  the  gelatin  or  agar- 
agar  is  quite  solid.  This  takes  longer  with  gelatin 
than  with  agar.  When  quite  solid  they  are  placed 
upon  little  glass  benches  (Fig.  13),  and  each  bench  is 

FIG.  13. 


labelled  with  the  number  of  the  plate  in  the  series  of 
dilutions.  The  benches,  with  the  plates  upon  them,  are 
then  piled  one  above  the  other  in  a  glass  dish,  the  so- 
called  "  culture-dish,"  in  which  the  plates  are  to  be  kept 
during  the  growth  of  the  bacteria.  The  benches  are 
sterilized  before  using,  in  the  way  given  for  the  plates. 

CULTURE-DISH. — This  dish,  which  is  about  22  cm.  in 
diameter  and  has  vertical  sides  of  about  6  cm.  in  height, 

5 


86  BACTERIOLOGY. 

is  provided  with  a  cover  of  exactly  the  same  design,  but 
of  a  little  larger  diameter.  This  cover,  when  placed 
upon  the  dish  containing  the  plates,  fits  over  it  and 
prevents  the  access  of  dust.  Prior  to  using,  the  dish  and 
cover  should  have  been  disinfected  for  one-half  hour  with 
1:1000  sublimate,  and  then  all  the  sublimate  solution 
allowed  to  drain  from  it. 

Into  the  bottom  of  this  dish  is  sometimes  placed  a  disc 
of  sterilized  filter-paper  moistened  with  sterilized  water, 
which  serves  to  prevent  the  drying  of  the  plates.  This, 
however,  is  not  necessary. 

If  agar-agar  is  employed,  the  dish  and  its  contents  may 
be  placed  at  a  temperature  of  37°-38°  C. ;  if  gelatin, 
the  temperature  at  which  the  plates  are  now  to  be  kept 
should  not  be  over  22°  C.,  otherwise  the  gelatin  becomes 
liquefied  and  the  plates  are  rendered  useless. 

When  development  has  occurred,  the  object  of  the 
dilution  will  easily  be  seen,  and  the  different  species  of 
bacteria  in  the  mixture  will  be  recognized  by  differences 
in  the  character  of  the  colonies  growing  from  them. 

This,  in  short,  is  the  plate  method  of  Koch  for  the 
separation  of  the  individual  species  contained  in  a  mix- 
ture of  bacteria.  Many  modifications  of  this  method 
exist ;  all,  however,  are  based  upon  the  same  principles. 
The  modifications  have  for  their  object  the  accomplish- 
ment of  the  same  end,  but  with  a  smaller  armamenta- 
rium of  apparatus. 

PETRI'S  MODIFICATION  OF  THE  PLATE  METHOD. — 
The  modification  which  approaches  nearest  to  the  original 
method,  and  at  the  same  time  lessens  very  materially  the 
number  of  steps  in  the  process,  is  that  suggested  by  Petri. 
It  consists  in  substituting  for  the  plates  small,  round, 
double  glass  dishes,  which  have  about  the  same  surface- 


ESMARCH'S  TUBES.  87 

area  as  the  plates.  The  liquid  medium  may  be  poured 
directly  into  these  little  dishes  without  their  being  exactly 
level.  Each  dish  acts  as  a  plate.  Their  covers  are  then 
to  be  replaced,  and  they  are  set  aside  for  observation. 
In  all  other  respects  the  steps  are  the  same  as  those  given 
for  Koch's  original  method.  Petri's  dishes  are  flat,  double 
dishes  of  glass  (Fig.  14).  They  are  of  about  8  cm.  in 

FIG.  14. 


diameter  and  about  1.5  to  2  cm.  in  height,  the  walls 
being  vertical.  They  may  readily  be  sterilized  either 
by  the  hot-air  or  steam  methods  of  sterilization.  They 
are  very  useful  for  this  work,  as  they  do  away  with  the 
necessity  for  the  cooling-stage  and  levelling  tripods, 
though  in  warm  weather  the  cooling-stage  may  be  used 
to  hasten  the  solidification  of  gelatin. 

ESMARCH'S  TUBES. — The  modification  of  Koch's 
method  which  insures  the  greatest  security  from  con- 
tamination by  outside  organisms  and  requires  the  small- 
est supply  of  apparatus,  is  that  suggested  by  v.  Esmarch. 
It  differs  from  the  other  methods  thus  :  The  dilutions 
having  been  prepared  in  tubes  containing  a  smaller 
amount  of  medium  than  usual — as  a  rule  not  more  than 
5  to  6  c.c. — are,  instead  of  being  poured  out  upon  plates 
or  into  dishes,  spread  over  the  inner  surface  of  the  tube 
containing  them,  and  without  removing  the  cotton  plugs 
are  caused  to  solidify  iu  this  position.  The  tubes  then 


88 


BACTERIOLOGY. 


present  a  thin  cylindrical  lining  of  gelatin  or  agar-a^ar. 
upon  which  the  colonies  develop.  In  all  other  respects 
the  conditions  for  the  growth  of  the  organisms  are  the 
same  as  in  flat  plates. 

Esmarch  directs  that  after  completion  of  the  dilutions 
the  tops  of  the  cotton  plugs  in  the  tubes  should  be  cut 
off  flush  with  the  mouth  of  the  test-tube  and  a  rubber 
cap  be  placed  over  this.  They  are  then  to  be  held  in 
the  horizontal  position  and  twisted  between  the  fingers 
upon  their  long  axes  under  ice-water.  The  gelatin  be- 
comes solidified  thereby  and  adheres  to  the  sides  of  the 
tube.  When  the  gelatin  is  quite  hard  the  tubes  are 
removed  from  the  water,  wiped  dry,  the  rubber  caps 
removed,  and  they  are  set  aside  for  observation. 

FIG.  15. 


For  some  time  past  we  have  deviated  from  the  direc- 
tions given  by  v.  Esmarch  for  this  part  of  his  method. 
Instead  of  rolling  the  tubes  under  ice-water,  we  roll 
them  upon  a  block  of  Ice  (Fig.  15).  In  this  method  a 
small  block  of  ice  only  is  needed.  It  is  arranged  nearly 


ESMARCH'S  TUBES.  89 

level,  and  is  held  in  position  by  being  placed  in  a  dish 
upon  cloths.  A  horizontal  groove  is  melted  in  the  sur- 
face of  the  ice  with  a  test-tube  of  hot  water.  The  tubes 
to  be  rolled  are  then  held  in  an  almost,  not  quite,  hori- 
zontal position  and  twisted  between  the  fingers  until  the 
sides  are  moistened  by  the  contents  to  within  about 

1  cm.  of  the  cotton  plug,  care  being  taken  that  the 
gelatin  does  not  touch  the  cotton ;  otherwise  the  latter 
becomes  adherent  to  the  sides  of  the  tube  and  is  difficult 
to  remove.     The  tube  is  then  placed  in  the  groove  in 
the  ice  and  rolled,  no  rubber  cap  or  cutting  off  of  the 
cotton  plug  being  necessary. 

The  advantages  of  this  process  over  that  followed  by 
v.  Esmarch  are  that  it  requires  less  time,  is  cleaner,  no 
rubber  caps  are  needed,  the  rolled  tubes  are  more  regu- 
lar, and  the  gelatin  does  not  touch  the  cotton  plug,  as 
is  always  the  case  in  the  tubes  rolled  under  water, 
because  of  the  impossibility  of  holding  them  steady  at 
one  level. 

There  is  an  impression  that  Esmarch  tubes  are  riot 
a  success  when  made  from  ordinary  nutrient  agar-agar 
because  of  the  tendency  of  this  medium  to  collapse  and 
fall  into  the  bottom  of  the  tube.  This  slipping  down 
of  the  agar-agar  is  due  to  the  water  that  is  squeezed 
from  it  during  solidification  getting  between  the  medium 
and  the  walls  of  the  tube.  This  can  easily  be  overcome 
by  allowing  the  rolled  tubes  to  remain  at  nearly  a  hori- 
zontal position,  the  cotton  end  of  the  tube  about  1.5  to 

2  cm.  higher  than  the  bottom  of  the  tube,  for  twenty  - 
four  hours  after  rolling  them.  During  this  time  the  edge 
of  the  agar-agar  nearest  the  cotton  plug  becomes  dried 
and  adherent  to  the  walls  of  the  tube,  while  the  water 
collects  at  the  most  dependent  point,  i.e.,  the  bottom  of  the 


90  BACTERIOLOGY. 

tube.  After  this  they  may  be  retained  in  the  upright 
position  without  fear  of  the  agar-agar  slipping  down. 
We  have  followed  this  process  for  several  years  with 
entire  satisfaction. 

In  all  these  processes,  if  the  dilutions  of  the  num- 
ber of  organisms  have  been  properly  conducted,  the 
results  will  be  the  same.  The  original  plate  or  tube, 
as  a  rule,  will  be  of  no  use  because  of  the  great  number 
of  colonies  contained  in  it.  Plate  or  tube  No.  2  may  be 
of  service,  but  plate  or  tube  3  will  usually  contain  the 
organisms  in  such  small  numbers  that  the  colonies 
originating  from  them  will  have  nothing  to  prevent 
their  characteristic  development. 

For  reasons  of  economy,  the  "original,"  tube  1, 
is  sometimes  substituted  by  a  tube  containing  normal 
salt  solution  (0.6  to  0.7  per  cent,  of  sodium  chloride 
in  water),  which  is  thrown  aside  as  soon  as  the  dilu- 
tions are  completed  and  only  plates  or  tubes  2  and  3 
are  made. 


CHAPTER   IX. 

The  incubator  used  in  bacteriological  work — Gas-pressure  regu- 
lator— Thermo-regulator — The  form  of  burner  employed  in  heating 
the  incubator. 

THE  INCUBATOK — When  the  plates  have  been  made, 
it  must  be  borne  in  mind  that  for  the  development  of 
certain  forms  of  bacteria  a  higher  temperature  is  neces- 
sary than  for  the  growth  of  others.  The  pathogenic 
or  disease-producing  organisms  all  grow  more  luxuri- 
antly at  the  temperature  of  the  human  body  (37.5°  C.) 
than  at  lower  temperatures ;  whereas,  with  the  ordinary 
saprophytic  forms  almost  any  temperature  between  18° 
to  2.0°  C.  and  that  of  the  body  is  favorable.  It  there- 
fore becomes  necessary  to  provide  some  place  in  which 
a  constant  temperature  suitable  to  the  growth  of  the 
pathogenic  organisms  can  be  maintained.  For  this 
purpose  there  have  been  devised  a  number  of  different 
forms  of  apparatus.  Fundamentally  they  are  all  based 
upon  the  same  principles,  however,  and  a  general  de- 
scription of  the  essential  points  involved  in  their  con- 
struction will  be  all  that  is  needed  here. 

This  apparatus  has  the  names  thermostat,  incubator, 
and  brooding  oven.  It  is  a  copper  chamber  (Fig.  16) 
with  double  walls,  the  space  between  which  is  filled  with 
water.  The  incubating  chamber  may  be  opened  or  closed 
by  a  closely  fitting  double  door,  inside  of  which  is  usually 
a  false  door  of  glass  through  which  the  contents  of  the 
chambers  may  be  inspected  without  actually  opening  it. 


92 


BACTERIOLOGY. 


The  whole  apparatus  is  encased  in  either  abestos  boards 
or  thick  felt  to  prevent  radiation  of  heat  and  consequent 
fluctuations  in  temperature.  In  the  top  of  the  chamber 


FIG.  16. 


is  a  small  opening  through  which  a  thermometer  pro- 
jects into  its  interior.  At  either  corner,  leading  into  the 
space  containing-  the  water  are  other  openings,  for  the 
reception  of  another  thermometer  and  a  thermo-regulator, 
and  for  refilling  the  apparatus  as  the  water  evaporates. 


THE    INCUBATOK. 


93 


On  the  side  is  a  water-gauge  for  showing  the  level  of 
the  water  between  the  walls.  The  object  of  the  water 
chamber,  which  is  formed  by  the  double  wall  arrange- 
ment, is  to  insure  by  means  of  the  warmed  water  an 
equable  temperature  at  all  parts  of  the  apparatus — at 
the  top  as  well  as  at  the  sides,  back,  and  bottom.  The 
apparatus  should  be  kept  filled  with  water,  otherwise 
the  object  for  which  it  is  constructed  will  not  be  accom- 
plished. When  the  chamber  between  the  walls  is  filled 
with  water  the  apparatus  is  heated  from  a  gas-flame 
which  is  placed  beneath  it. 

FIG.  17. 


The  burner  employed  in  heating  the  incubator  is 
known  as  "  Koch's  safety  burner"  (Fig.  17).  It  is  a 
Bunsen  burner  provided  with  an  arrangement  for  auto- 
matically turning  off  the  gas  supply  and  thus  preventing 


94  BACTERIOLOGY. 

accidents  should  the  flame  become  extinguished  at  a  time 
when  no  one  was  near.  The  gas-cock  is  provided  with 
a  long  arm  which  is  weighted  and  which,  when  the  gas 
is  turned  on  and  burning,  rests  upon  the  end  of  a  metal 
spiral  which  is  heated  by  the  flame.  If  by  draughts  or 
any  other  accident  the  flame  becomes  extinguished  the 
metal  spiral  cools,  arid  in  cooling  contracts  and  allows 
the  weighted  arm  of  the  gas-cock  to  fall.  By  its  falling 
the  gas  supply  is  turned  off. 

THERMO-REGULATORS. — The  regulation  and  mainte- 
nance of  the  proper  temperature  within  the  incubator 
is  accomplished  by  the  employment  of  an  automatic 
thermo- regulator. 

The  form  of  thermo-regulator  used  for  this  purpose 
is  constructed  upon  principles  involving  the  expansion 
and  contraction  of  fluid  substances  under  the  influence 
of  heat  and  cold.  By  means  of  this  expansion  and  con- 
traction, the  amount  of  gas  passing  from  the  source  of 
supply  to  the  burner  may  be  either  diminished  or  in- 
creased as  the  temperature  of  the  substance  in  which  the 
regulator  is  placed  either  rises  or  falls. 

The  simplest  form  of  thermo-regulator  which  serves 
to  illustrate  the  principles  involved  is  seen  in  Fig.  18. 

It  consists  of  a  glass  cylinder  e,  having  a  communicat- 
ing branch  tube  6,  and  rubber  stoopper  /,  through 
which  projects  the  bent  tube  a.  The  tube  a  is  ground 
to  a  slanting  point  at  the  extremity  which  projects  into 
the  tube  et  and  is  provided  a  short  distance  above  this 
point  with  a  capillary  opening  g,  in  one  of  its  sides. 

When  ready  for  use  the  cylinder  e  is  filled  with  fluid, 
either  mercury,  calcium  chloride  solution,  alcohol  and 
ether,  or  a  number  of  other  substances  depending  upon 
the  temperature  to  which  it  is  to  be  subjected,  up  to  about 


THERMO-REGULATORS. 


95 


the  level  shown  in  the  cut.  It  is  then  allowed  to  stand 
or  is  suspended  in  the  bath  the  temperature  of  which  it 
is  to  regulate.  The  rubber  tubing  coming  from  the  gas 
supply  is  attached  to  the  outer  end  of  the  glass  tube  a,  and 

FIG.  18. 


the  tube  going  to  the  burner  is  slipped  over  the  branch 
tube  b.  The  gas  is  turned  on  and  the  burner  lighted 
and  placed  under  the  bath.  The  gas  now  streams 
through  the  tube  a  into  the  cylinder  e  and  out  at  b 
to  the  burner,  but  as  the  temperature  of  the  bath 
rises,  the  fluid  contained  in  the  cylinder  e,  under 


96  BACTERIOLOGY. 

the  influence  of  the  elevation  of  temperature  begins  to 
expand,  and  as  a  continuous  rise  in  temperature  pro- 
ceeds, the  expansion  of  the  fluid  accompanies  it  and 
gradually  closes  the  slanting  opening  h  of  tube  a.  In 
this  way  the  supply  of  gas  becomes  diminished  and 
the  rise  in  temperature  of  the  bath  will  be  less  rapid, 
until  finally  the  opening  at  h  will  be  closed  entirely, 
when  the  supply  of  gas  to  the  burner  will  now  be 
limited  to  that  passing  through  the  capillary  opening 
g.  This  is  not  sufficient  to  maintain  the  highest  tem- 
perature reached,  and  a  gradual  contraction  of  the 
fluid  now  occurs  until  there  is  again  an  outflow  of  gas 
from  the  opening  h,  when  again  the  temperature  rises. 
This  contraction  and  expansion  of  the  fluid  in  the  regu- 
lator continues  until  eventually  a  point  is  reached  at 
which  the  position  of  the  fluid  in  the  cylinder  e  allows 
of  the  passage  of  just  enough  gas  from  the  opening  h  to 
maintain  a  constant  temperature.  This,  in  short,  is  the 
principle  on  which  thermo-regulators  are  constructed, 
but  it  must  be  borne  in  mind  that  a  great  deal  of  detail 
exists  in  the  construction  of  an  accurate  instrument. 
The  number  of  different  forms  of  this  apparatus  is  com- 
paratively large,  and  form  has  each  its  special  merits. 

The  value,  that  is,  the  delicacy  of  the  thermo-regulator 
depends  upon  a  number  of  factors,  all  of  which  it  would 
be  useless  to  introduce  into  a  book  of  this  kind,  but  in 
general  it  may  be  said  that  the  essential  points  to  be 
observed  in  selecting  a  thermo-regulator  depend  in  the 
main  upon  the  temperatures  to  which  it  is  to  be  applied. 
For  low  temperatures  such  fluids  as  ether,  alcohol,  and 
calcium  chloride  solution,  which  expand  and  contract 
rapidly  and  regularly  under  slight  variations  in  temper- 
ature, are  commonly  employed ;  whereas  for  temperatures 


GAS-PRESSURE    REGULATORS. 


97 


approaching  the  boiling-point  of  water  mercury  is  most 
frequently  used. 

The  temperature  of  the  incubator  is  to  be  regulated, 
then,  by  the  use  of  some  such  form  of  apparatus  as  that 
just  described.  It  should  be  of  sufficient  delicacy  to 
prevent  a  fluctuation  of  more  than  0.2°  C.  in  the  tem- 
perature of  the  air  within  the  chamber  of  the  apparatus. 


G.  19. 


GAS-PRESSURE  REGULATORS. — A  gas-pressure  regu- 
lator is  not  rarely  intervened  between  the  gas  supply 
and  the  thermo-regulator.  This  apparatus  has  for  its 
object  the  maintenance  of  a  constant  pressure  of  the  gas 
going  to  the  thermo-regulator.  There  are  several  in- 
struments of  this  form  in  use,  but  none  of  them  accom- 
plishes the  object  for  which  they  are  designed. 


98  BACTERIOLOGY. 

The  instrument  most  commonly  employed,  the  appar- 
atus of  Moitessier  (Fig.  19),  is  based  on  somewhat  the 
same  principles  as  the  large  regulators  seen  at  the  manu- 
factories of  illuminating  gas,  which  act  very  well  when 
employed  on  the  large  scale,  as  one  sees  them  there ; 
but  which,  when  applied  to  the  limited  and  sudden  fluc- 
tuations seen  in  the  gas  coming  from  an  ordinary  gas- 
cock,  are  practically  useless.  They  are  too  gross  in  their 
construction,  and  are  only  seen  to  act  tinder  compara- 
tively great  and  gradual  fluctuations  in  pressure.  If  a 
good  form  of  thermo-regulator  is  employed,  there  is  no 
necessity  for  the  use  of  any  of  the  forms  of  pressure- 
regulators  thus  far  introduced. 


CHAPTER    X. 

The  study  of  colonies— Their  naked-eye  peculiarities  and  their 
appearance  under  different  conditions — Differences  in  the  structure 
of  colonies  from  different  species  of  bacteria — Stab  cultures— Slant 
cultures. 

THE  plates  upon  agar-agar  which  have  been  prepared 
from  a  mixture  of  organisms  and  have  been  placed  in 
the  incubator,  and  those  of  gelatin  which  have  been  main- 
tained at  the  ordinary  temperature  of  the  room,  are 
usually  ready  for  examination  after  twenty- four  to 
forty-eight  hours.  They  will  be  found  to  be  marked 
here  and  there  by  small  points  or  little  islands  of  more 
or  less  opaque  appearance.  In  some  instances  these 
will  be  so  transparent  that  it  is  with  difficulty  one  can 
see  them  with  the  naked  eye.  Again,  they  may  be  of  a 
dense  opaque  appearance,  at  one  time  sharply  circum- 
scribed and  round,  again  irregular  in  their  outline. 
Here  a  point  will  present  one  color,  there  perhaps 
another.  On  gelatin  some  of  the  points  will  be  seen  to 
be  lying  on  the  surface  of  the  medium,  while  others  will 
be  in  the  centre  of  a  slight  depression,  the  result  of 
liquefaction  of  the  gelatin  about  this  point. 

Place  the  plate  containing  these  points  upon  the  stage 
of  the  microscope  and  examine  them  with  the  lowest 
power  objective,  and  again  differences  will  be  observed. 
Some  of  these  minute  points  will  be  finely  granular, 
others  coaVsely  so ;  some  will  present  a  radiated  appear- 
ance, while  a  neighbor  may  be  concentrically  arranged. 


100  BACTERIOLOGY. 

Here  nothing  particularly  characteristic  will  present, 
there  the  point  may  resolve  itself  into  a  little  mass  hav- 
ing somewhat  the  appearance  of  a  very  small  pellicle  of 
raw  cotton.  All  these  differences,  and  many  more,  aid 
us  in  saying  that  these  little  points  must  be  different 
in  their  nature.  With  a  pointed  platinum  needle  take  up 
a  bit  of  one  of  these  little  islands,  prepare  a  stained  cover- 
slip  preparation  (see  chapter  on  cover-slip  preparations) 
from  it,  and  examine  it  under  the  high  power  oil-immer- 
sion objective,  under  access  of  the  greatest  amount  of 
light  aiforded  by  the  illuminator  of  the  microscope.  The 
preparation  will  be  seen  to  be  made  up  entirely  of  bodies 
of  the  same  shape ;  they  will  all  be  spheres,  or  ovals,  or 
rods,  but  not  a  mixture  of  these  forms,  if  proper  care  in 
the  manipulation  has  been  taken.  Examine  in  the  same 
way  a  neighboring  spot  which  possesses  different  naked- 
eye  appearances,  and  it  will  be  found  to  consist  of  bodies 
of  an  entirely  different  appearance  from  those  in  the 
first  preparation. 

These  spots  or  islands  on  the  surface  of  the  plates  are 
colonies  of  bacteria,  differing  severally,-  not  only  in  out- 
ward appearances,  the  one  from  the  other,  but,  as  our 
cover-slip  preparations  show,  in  the  morphological  char- 
acteristics of  the  individual  organisms  composing  them. 
If  from  one  of  these  colonies  a  second  set  of  plates  be 
prepared,  the  peculiarities  which  were  at  first  observed  in 
this  colony  will  be  reproduced  in  the  new  set  of  colonies 
which  develop.  In  other  words,  these  peculiarities  are 
constant  under  constant  conditions.  The  colonies  will 
be  found  to  consist  of  the  same  organisms  as  the  colony 
from  which  the  plates  were  made,  and  colonies  of  no 
other  organisms  will  be  present. 

With  all  organisms  differences  in  the  appearance  of 


STAB    AND    SMEAR    CULTURES.  101 

the  colonies  depending  upon  their  location  in  the  medium 
can  usually  be  detected.  When  deep  down  in  the  medium, 
owing  to  surrounding  pressure,  they  are  quite  round, 
oval,  or  lozenge-shape ;  whereas,  when  they  are  on  the 
surface  of  the  gelatin  or  agar,  they  may  take  quite  a 
different  form.  This  is  purely  a  mechanical  effect,  and 
is  always  to  be  borne  in  mind,  otherwise  errors  are 
apt  to  arise. 

PURE  CULTURES. — If  from  one  of  these  small  colonies 
a  bit  be  taken  upon  the  point  of  a  sterilized  platinum 
needle  and  introduced  into  a  tube  of  sterilized  gelatin 
or  agar-agar,  the  growth  that  results  will  be  what  is 
known  as  a  "  pure  culture,77  the  condition  in  which  all 
organisms  must  be  before  a  systematic  study  of  their 
many  pecularities  is  begun.  Sometimes  several  series  of 
plates  are  necessary  before  the  organism  can  be  obtained 
pure,  but  by  patiently  following  this  plan  the  results 
will  ultimately  be  satisfactory. 

TEST-TUBE  CULTURES;  STAB  CULTURES;  SMEAR 
CULTURES. — After  separating  the  organisms,  the  one 
from  the  other  by  the  plate  method  just  described,  they 
must  be  isolated  from  the  plates  as  pure  stab  or  smear 
cultures. 

This  is  done  in  the  following  way  :  Decide  upon  the 
colony  from  which  the  pure  culture  is  to  be  made.  Select 
preferably  a  small  colony  and  one  as  widely  separated 
from  other  colonies  as  possible.  Sterilize  in  the  gas- 
flame  a  straight  platinum-wire  needle.  The  glass  handle 
of  the  needle  should  be  drawn  through  the  flame  as  well 
as  the  needle  itself,  otherwise  contamination  from  this 
source  may  occur.  When  it  is  cool,  which  is  in  three 
to  five  seconds,  take  up  carefully  a  portion  of  the  colony. 
Guard  against  touching  anything  but  the  colony.  If, 


102  BACTERIOLOGY. 

during  manipulation,  the  needle  touches  anything  else 
whatever  than  the  colony  from  which  the  culture  is  to 
be  made,  it  must  be  sterilized  again.  This  holds  not 
only  for  the  time  before  touching  the  colony,  but  also 
during  its  passage  into  the  test-tube  from  the  colony, 
otherwise  there  is  no  guarantee  that  the  growth  resulting 
from  the  inoculation  of  this  bit  of  colony  into  a  fresh 
sterile  medium  will  be  pure. 

In  the  meantime  have  in  the  other  hand  a  test-tube 
of  sterile  medium  :  gelatin,  agar-agar,  or  potato.  This 
tube  is  held  across  the  palm  of  the  hand  in  an  almost 
horizontal  position  with  its  mouth  pointing  out  between 
the  thumb  and  index  finger  and  its  contents  toward 
the  body  of  the  worker.  With  the  disengaged  fingers 
of  the  hand  holding  the  needle,  the  cotton  plug  is  removed 
from  the  tube  by  a  twisting  motion  and  placed  between 
the  index  and  second  fingers  of  the  hand  holding  the 
tube  in  such  a  way  that  the  portion  of  the  plug  which 
fits  into  the  mouth  of  the  test-tube  looks  toward  the 
dorsal  surface  of  the  hand  and  does  not  touch  any  portion 
of  the  hand — this  is  accomplished  by  placing  only  the  over- 
hanging portion  of  the  plug  between  the  fingers.  The 
needle  containing  the  bit  of  colony  is  now  to  be  thrust 
into  the  medium  in  the  tube  if  a  stab  culture  is  desired, 
or  rubbed  gently  over  its  surface  if  a  smear  culture  is  to 
be  made.  The  needle  is  then  withdrawn,  the  cotton  plug 
replaced,  and  the  needle  sterilized  before  it  is  laid  down. 
Neither  the  needle  nor  its  handle  should  touch  the 
inner  sides  of  the  test-tube  if  it  can  be  avoided. 

The  tube  is  then  labelled  and  set  aside  for  observa- 
tion. The  growth  which  appears  in  the  tube  after 
twenty-four  to  thirty-six  hours  will  be  a  pure  culture 
of  the  organisms  of  which  the  colony  was  composed. 


STAB    AND    SMEAK    CULTURES.  103 

Cultures  of  this  form  are  not  only  useful  as  a  means 
of  preserving  pure  cultures  of  the  different  organisms 
with  which  we  may  be  working,  but  serve  also  to  bring 
out  certain  characteristics  of  different  organisms  when 
grown  in  this  way. 

If  gelatin  is  employed  and  the  organism  which  has 
been  introduced  into  it  possesses  the  power  of  bringing 
about  liquefaction,  this  result  is  by  no  means  of  the  same 
appearance  for  all  organisms.  Some  organisms  cause  a 
liquefaction  which  spreads  across  the  whole  upper  sur- 
face of  the  gelatin  and  continues  gradually  downward  ; 
again  it  occurs  in  a  funnel  shape,  the  broad  end  of  the 
funnel  being  uppermost  and  the  point  downward,  corre- 
sponding to  the  track  of  the  needle.  At  times  a  stock- 
ing- or  sac-formed  liquefaction  may  be  noticed. 

Obtain  a  number  of  organisms  from  different  sources 
in  pure  cultures  by  the  method  given.  Plant  them  as 
pure  cultures,  all  at  the  same  time,  in  gelatin — preferably 
gelatin  of  the  same  making — retain  them  under  the  same 
conditions  of  temperature,  and  note  the  finer  differences 
in  the  way  in  which  liquefaction  occurs. 


CHAPTER   XI. 

Systematic  study  of  an  organism — Steps  necessary  in  identifying 
an  organism  as  a  definite  species. 

AFTER  isolating  an  organism  by  the  plate  method, 
considerable  work  is  necessary  in  order  to  establish  its 
identity  as  a  definite  species. 

It  must  possess  certain  morphological  and  cultural 
peculiarities,  which  must  be  constant  under  constant  con- 
ditions. 

Its  form  at  certain  stages  must  always  be  the  same. 
Its  ability  or  inability  to  produce  spores  must  not  vary 
under  proper  conditions.  Its  growth  upon  the  different 
media  under  constant  conditions  of  temperature  must 
always  present  the  same  outward  appearances.  The 
reactions  given  by  it  to  the  media  in  which  it  is  growing 
must  follow  a  fixed  rule.  Its  power  to  produce  liquefac- 
tion of  the  gelatin,  or  to  grow  upon  it  without  bringing 
about  this  change,  must  always  be  the  same.  Its  motility 
or  non-motility  must  be  determined.  Its  production  of 
certain  chemical  products  must  be  detected  by  chemical 
analysis.  Its  behavior  toward  oxygen — i.  e.,  does  it  re- 
quire this  gas  for  its  growth  ?  is  this  gas  an  indifferent 
factor?  or  by  its  presence  are  the  life  processes  of  the 
organism  checked? — must  be  determined.  Its  behavior 
under  varying  conditions  of  temperature  and  under  the 
influence  of  different  chemical  bodies  as  well  as  its  growth 
in  media  of  different  reactions  are  to  be  studied.  The 
property  of  producing  fermentation  with  the  libera- 


MICROSCOPIC    EXAMINATIONS.  105 

tion  of  gases  must  be  ascertained ;  and  lastly,  we  must 
consider  its  behavior  when  introduced  into  the  bodies  of 
animals  used  for  experimental  work — i.  e.,  is  it  a  disease- 
producing  organism,  or  does  it  belong  to  the  group  of 
innocent  saprophytes  ? 

We  have  learned  the  methods  for  obtaining  colonies, 
and  have  studied  some  of  the  peculiarities  which  are  to 
distinguish  them  from  one  another.  The  next  important 
step  is  to  determine  the  morphology  of  the  individuals 
composing  these  colonies  as  well  as  their  relation  to  each 
other  in  the  colony.  These  points  are  decided  by  micro- 
scopic examination  of  bits  of  the  colony  which  have  been 
transferred  to  thin  glass  cover-slips,  upon  which  they  are 
dried,  stained,  and  mounted.  Cover-slips  for  this  pur- 
pose are  prepared  in  two  ways  :  either  by  taking  up  a 
bit  of  the  colony  on  a  needle,  smearing  it  upon  a  cover- 
slip,  staining  it,  and  examining  it — by  which  only  the 
morphology  of  the  individuals  can  be  made  out — or  by 
the  method  of  "impression  cover- slip  preparations,"  by 
which  not  only  the  morphology,  but  also  the  relation  of 
the  organisms  to  one  another  in  the  colony  can  be  deter- 
mined. The  details  of  these  methods  will  be  found  in 
the  chapter  on  the  methods  of  staining. 

MICROSCOPIC  EXAMINATION  OF  PREPARATIONS. 

THE  DIFFERENT  PARTS  OF  THE  MICROSCOPE. — 
Before  describing  the  process  of  examining  preparation? 
microscopically,  a  few  definitions  of  the  terms  used  in 
referring  to  the  microscope  may  not  be  out  of  place. 

The  ocular  or  eye-piece  is  the  lens  at  which  the  eye 
is  placed  in  looking  through  the  instrument. 

The  objective  is  the  lens  which  is  at  the  distal  end  of 


106  BACTERIOLOGY. 

the  barrel  of  the  instrument,  and  which  serves  to  mag- 
nify the  object  to  be  examined. 

The  stage  is  the  shelf  or  platform  of  the  microscope 
on  which  the  object  rests. 

The  reflector  is  the  mirror  placed  beneath  the  stage, 
which  serves  to  direct  the  light  to  the  object  to  be 
examined. 

The  coarse  adjustment  is  the  rack-and-pinion  arrange- 
ment by  which  the  barrel  of  the  microscope  can  be 
quickly  raised  or  lowered. 

The  fine  adjustment  serves  to  raise  and  lower  the 
barrel  of  the  instrument  very  slowly  and  gradually. 

For  the  microscopic  study  of  bacteria  it  is  essential 
that  the  microscope  be  provided  with  an  oil-immersion 
system  and  a  sub-stage  condensing  apparatus. 

The  Oil-immersion  System  consists  in  an  objective  so 
constructed  that  it  can  only  be  used  when  the  media 
through  which  the  light  passes  in  entering  it  are  all  of 
the  same  index  of  refraction — i.  e.,  are  homogeneous. 
This  is  accomplished  by  interposing  between  the  face  of 
the  lens  and  the  cover-slip  covering  the  object  to  be  ex- 
amined a  body  which  refracts  the  light  in  the  same 
way  as  do  the  glass  slide,  the  cover-slip,  and  the  glass 
of  which  the  objective  is  made.  For  this  purpose  a 
drop  of  oil  of  the  same  index  of  refraction  as  the  glass 
is  placed  upou  the  face  of  the  lens,  and  the  examina- 
tions are  made  through  this  oil.  There  Fs  thus  no  loss 
of  light  from  deflection,  as  is  the  case  in  the  dry  systems. 

The  sub-stage  condensing  apparatus  is  a  system  of 
lenses  situated  beneath  the  central  opening  of  the  stage. 
They  serve  to  condense  the  light  passing  from  the 
reflector  to  the  object  in  such  a  way  that  it  is  focussed 
upon  the  object.  Between  the  condenser  and  reflector 


MICROSCOPIC    EXAMINATIONS.  107 

is  placed  an  adjustable  diaphragm,  the  aperture  of  which 
can  be  regulated,  as  circumstances  require,  to  permit  of 
either  a  very  small  or  very  large  amount  of  light  passing 
to  the  object. 

MICROSCOPIC  EXAMINATION  or  COVER-SLIPS. — 
The  stained  cover-slip  is  to  be  examined  with  the  oil- 
immersion  objective,  and  with  the  diaphragm  of  the  sub- 
stage  condensing  apparatus  open  to  its  full  extent.  The 
object  gained  by  allowing  the  light  to  enter  in  such 
a  large  volume  is  that  the  contrast  produced  by  the 
colored  bacteria  in  the  brightly  illuminated  field  is  much 
more  conspicuous  than  when  a  smaller  amount  of  light 
is  thrown  upon  them.  This  is  true  not  only  for  bacteria 
on  cover-slips,  but  likewise  for  their  differentiation  from 
surrounding  objects  when  they  are  located  in  tissues. 
With  unstained  bacteria  and  tissues,  on  the  contrary, 
the  structure  can  best  be  made  out  by  reducing  the 
bundle  of  light-rays  to  its  smallest  amount,  and  in  this 
way  favoring,  not  color  contrast,  but  contrasts  which 
appear  as  lights  and  shadows  due  to  the  differences  in 
permeability  to  light  of  the  various  parts  of  the  material 
under  examination. 

STEPS  IN  EXAMINING  STAINED  PREPARATIONS 
WITH  THE  OIL-IMMERSION  SYSTEM. — Place  upon  the 
centre  of  the  cover-slip  which  covers  the  preparation  a 
small  drop  of  immersion  oil.  Place  the  slide  upon  the 
centre  of  the  stage  of  the  microscope.  With  the  coarse 
adjustment  lower  the  oil-immersion  objective  until  it 
just  touches  the  drop  of  oil.  Open  the  illuminating  appa- 
ratus to  its  full  extent.  Then,  with  the  eye  to  the  micro- 
scope and  the  hand  on  the  fine  adjustment,  turn  the 
adjusting-screw  toward  the  right  until  the  field  becomes 
somewhat  colored  in  appearance.  When  this  is  seen, 


108  BACTERIOLOGY. 

proceed  more  slowly  in  the  same  direction,  and,  after  one 
or  two  turns,  the  object  will  be  in  focus.  Do  not  remove 
the  eye  from  the  instrument  until  this  has  been  accom- 
plished. 

Then,  with  one  hand  upon  the  fine  adjustment  and 
the  thumb  and  index  finger  of  the  other  hand  holding 
lightly  the  slide  by  its  end,  the  slide  may  be  moved 
about  under  the  objective.  At  the  same  time  the  screw 
of  the  fine  adjustment  must  be  turned  back  and  forth  so 
that  the  different  levels  of  the  preparation  may  one  after 
the  other  be  brought  into  focus.  In  this  way  the  whole 
section  or  preparation  may  be  inspected.  When  the 
examination  is  finished,  raise  the  objective  from  the 
preparation  by  turning  the  screw  of  the  coarse  adjust- 
ment toward  you.  Remove  the  preparation  from  the 
stage,  and,  with  a  fine  silk  cloth  or  handkerchief,  wipe 
very  gently  and  carefully  the  oil  from  the  face  of  the  lens. 
The  lens  is  then  unscrewed  from  the  microscope  and 
placed  in  the  case  intended  for  its  reception. 

During  work,  of  course,  the  lens  need  not  be  cleaned 
and  put  away  after  each  examination  ;  but  when  the 
work  for  the  day  is  over,  an  immersion  lens  must  always 
be  protected  in  this  way.  Under  no  circumstances  should 
it  be  allowed  to  remain  in  the  immersion  oil  or  exposed 
to  dust  for  any  length  of  time. 

EXAMINATION  OF  UNSTAINED  PREPARATIONS. — 
" Hanging  drops"  It  frequently  becomes  necessary  to 
examine  bacteria  in  the  unstained  condition.  The  circum- 
stances calling  for  this  arise  while  studying  the  multipli- 
cation of  cells,  the  germination  of  spores,  the  formation 
of  spores,  and  the  absence  or  presence  of  motility. 

In  this  method  the  organisms  to  be  studied  are  sus- 
pended in  a  drop  of  salt  solution  or  bouillon  in  the 


MICROSCOPIC    EXAMINATIONS.  109 

centre  of  a  cover-slip.  This  is  then  placed,  drop  down, 
upon  an  object-glass  in  the  centre  of  which  a  hollow  or 
depression  is  ground  (Fig.  20).  The  slip  is  held  in  posi- 
tion by  a  thin  layer  of  vaselin,  which  may  be  painted 

FIG.  20. 


around  the  margins  of  the  depression.  This  not  only 
prevents  the  slip  from  moving  from  its  position  during 
examination,  but  also  prevents  drying  by  evaporation  if 
the  preparation  is  to  be  observed  for  any  length  of 
time.  This  is  known  as  the  "  hanging  drop  "  method  of 
examination  or  cultivation.  It  is  indispensable  for  the 
purposes  mentioned,  and  at  the  same  time  requires  con- 
siderable care  in  its  manipulation.  The  fluid  is  so  trans- 
parent that  the  cover- slip  is  often  broken  and  the  face 
of  the  objective  injured  by  its  being  brought  down  upon 
the  preparation  before  one  is  aware  that  the  focal  dis- 
tance has  been  reached.  This  may  be  avoided. by  grasp- 
ing the  slide  with  the  left  hand  and  moving  it  back 
and  forth  under  the  objective  as  it  is  brought  down 
toward  the  object.  As  soon  as  the  least  pressure  is  felt 
upon  the  slide  the  objective  must  be  raised,  otherwise 
the  cover-slip  will  be  broken  and  the  lens  may  be  ren- 
dered worthless. 

A  safer  plan  is  to  bring  the  edge  of  the  drop  into  the 
centre  of  the  field  with  one  of  the  higher  power  dry 
lenses.  When  this  is  accomplished,  substitute  the  im- 
mersion for  the  dry  system,  and  the  edge  of  the  drop 
can  now  easily  be  found. 

6 


110  BACTERIOLOGY. 

In  examining  bacteria  by  this  method  there  is  a  pos- 
sibility of  error  that  must  be  guarded  against.  All 
microscopic  insoluble  particles  in  suspension  in  fluids 
possess  a  peculiar  tremor  or  vibratory  motion,  the  so- 
called  u  Brownian  motion."  This  is  very  apt  to  give 
the  impression  that  the  organisms  under  examination 
are  motile  when  in  truth  they  are  not  so,  their  move- 
ment in  the  fluid  being  due  only  to  this  molecular 
tremor. 

The  difference  between  the  motion  of  bodies  which  are 
undergoing  this  molecular  tremor  and  that  possessed  by 
certain  living  bacteria  is  that  the  former  particles  never 
move  from  their  place  in  the  field,  while  the  living 
bacteria  alter  their  position  in  relation  to  the  surround- 
ing organisms,  and  may  dart  from  one  position  in  the 
field  to  another.  With  some  cases  the  true  movement 
of  bacteria  is  very  slow  and  undulating,  while  in  others 
it  is  rapid  and  darting.  The  molecular  tremor  may  be 
seen  with  non-motile  and  with  dead  organisms. 

Prepare  three  hanging-drop  preparations,  one  from 
a  drop  of  dilute  India  ink,  a  second  from  a  culture  of 
micrococci,  and  a  third  from  a  culture  of  the  bacillus  of 
typhoid  fever.  In  what  way  do  they  differ  ? 

STUDY  OF  SPORE-FORMATION. — The  hanging-drop 
method  just  mentioned  is  not  only  employed  for  the  de- 
tection of  the  motility  of  an  organism,  but  for  the  study 
of  its  spore- forming  properties. 

Since  with  aerobic  organisms  spore-formation  occurs, 
as  a  rule,  only  in  the  presence  of  oxygen,  and  is  induced 
more  by  limitation  of  the  nutrition  of  the  organisms 
than  by  any  other  factor,  it  is  essential  that  these  two 
points  should  be  borne  in  mind  in  preparing  the  drop 
cultures  in  which  the  process  is  to  be  studied.  For 


STUDY    OF    SPORE-FORMATION.  Ill 

this  reason  the  drop  of  bouillon  should  be  small  and 
the  air-chamber  relatively  large. 

The  cover- slip  and  hollow-ground  slide  should  be 
carefully  sterilized,  and  with  a  sterilized  platinum  loop  a 
very  small  drop  of  bouillon  is  placed  in  the  centre  of 
the  cover-slip.  The  slip  is  then  inverted  over  the  hollow 
depression  in  the  sterilized  object-glass  and  sealed  with 
vaselin.  The  most  convenient  method  of  performing 
this  last  step  in  the  process  is  to  paint  a  ring  of  vaselin 
around  the  edges  of  the  hollow  in  the  slide,  and  then, 
without  taking  the  cover-slip  up  from  the  table  upon 
which  it  rests,  invert  the  hollow  over  the  drop  and  press 
it  gently  down  upon  the  cover-slip.  The  vaselin  causes 
the  slip  to  adhere  to  the  slide,  so  that  it  can  be  easily 
taken  up.  The  drop  now  hangs  in  the  centre  of  the 
small  air-tight  chamber  which  exists  between  the  de- 
pression in  the  slide  and  the  cover-slip.  (See  Fig.  20.) 

A  drop  of  sterilized  agar-agar  may  be  substituted  for 
the  bouillon.  It  serves  to  retain  the  organisms  in  a  fixed 
position,  and  the  process  may  be  more  easily  followed. 

As  soon  as  finished,  the  preparation  is  to  be  examined 
microscopically,  and  the  condition  of  the  organisms 
noted.  It  is  then  to  be  retained  in  a  warm  chamber 
especially  devised  for  the  purpose  and  kept  under  con- 
tinuous observation.  The  form  of  chamber  best  adapted 
for  the  purpose  is  one  which  envelops  the  whole  micro- 
scope. It  is  provided  with  a  window  through  which  the 
light  enters,  and  an  arrangement  for  moving  the  slide 
about  from  the  outside.  The  formation  of  spores  requires 
a  much  longer  time  than  the  germination  of  spores  into 
bacilli,  but  with  patience  both  processes  may  be  satis- 
factorily observed. 

It  will  be  noticed  that  the  description  of  this  process 


112  BACTERIOLOGY. 

is  very  much  like  that  which  has  just  been  given,  but 
differs  from  it  in  one  respect,  viz.,  that  in  this  manip- 
ulation we  are  not  making  a  preparation  which  is  simply 
to  be  examined  and  then  thrown  aside,  but  it  is  an 
actual  pure  culture,  and  must  be  kept  as  such,  other- 
wise the  observation  will  be  worthless.  For  this  reason 
the  greatest  care  must  be  observed  in  the  sterilization  of 
all  objects  employed.  Studies  upon  spore-formation  by 
this  method  frequently  continue  over  hours,  and  some- 
times days,  and  contamination  must,  therefore,  be  care- 
fully guarded  against.  The  study  should  be  begun  with 
the  vegetative  form  of  the  organism ;  the  hanging-drop 
preparation  should,  for  this  reason,  always  be  made  from 
a  perfectly  fresh  culture  of  the  organism  under  consider- 
ation, before  time  has  elapsed  for  spores  to  form. 

The  simple  detection  of  the  presence  or  absence  of 
spore-formation  can  in  many  cases  be  made  by  other 
methods.  For  example,  many  species  of  bacteria  which 
possess  this  property  form  spores  most  readily  upon 
media  from  which  it  is  somewhat  difficult  for  them  to 
obtain  the  necessary  nutrition;  potatoes  and  agar  agar 
which  have  become  a  little  dry  offer  very  favorable 
conditions,  because  of  the  limited  area  from  which  the 
growing  bacteria  can  draw  their  nutritive  supplies  and 
because  of  the  free  access  which  they  have  to  oxygen  ;  for, 
their  growth  being  on  the  surface,  they  are  surrounded 
by  this  gas  unless  means  are  taken  to  prevent  it.  By  the 
hanging-drop  method,  however,  more  than  this  simple 
property  may  be  determined.  It  is  possible  not  only 
to  detect  the  stages  and  steps  in  the  formation  of  en- 
dogenous spores,  but  when  the  spores  are  completely 
formed  by  transferring  them  to  a  fresh  bouillon-drop  or 
drop  of  agar-agar,  preserved  in  the  same  way,  their  ger- 


STUDY  OF  CULTURES  ON  POTATO.   113 

mination  into  mature  rods  may  be  seen.  The  word 
rods  is  used  because  as  yet  we  have  no  evidence  that 
endogenous  spore-formation  occurs  in  any  of  the  other 
morphological  groups  of  bacteria. 

STUDY  OF  GELATIN  CULTURES. — As  has  been  pre- 
viously stated,  the  behavior  of  bacteria  toward  gelatin 
differs — some  of  them  producing  apparently  no  altera- 
tion in  the  medium,  while  others  bring  about  a  form 
of  peptonization  which  results  in  liquefaction  of  the 
gelatin  at  and  around  the  place  at  which  the  colonies 
are  growing.  In  some  instances  this  liquefaction  spreads 
laterally,  in  others  it  sinks  directly  down  into  the  gelatin, 
These  differences  have  been  conspicuously  shown  and  em- 
ployed as  one  of  the  means  of  differentiation  of  otherwise 
closely  allied  members  of  the  same  family  of  bacteria. 
Studies  upon  the  organism  of  Asiatic  cholera  and  a 
number  of  closely  allied  forms  reveal  a  decided  differ- 
ence in  the  manner  of  liquefaction  produced  by  these 
different  organisms.  The  slightest  detail  in  this  respect 
must  be  noted,  and  its  frequency  or  constancy  under 
different  conditions  determined. 

CULTURES  ON  POTATO. — A  very  important  feature 
in  the  study  of  an  organism  is  its  growth  on  sterilized 
potato.  Many  organisms  present  appeal  anas  under 
this  method  of  cultivation  which  alone  can  almost  be 
considered  characteristic.  In  some  cases  coarsely  lobu- 
lated,  elevated,  dry  or  moist  patches  of  development 
occur  after  a  few  hours  ;  again,  the  growth  may  be  finely 
granular  and  but  slightly  elevated  above  the  surface  of 
the  potato ;  at  one  time  it  will  be  dry  and  dull  in 
appearance,  again  it  may  be  moist  and  glistening. 
Sometimes  there  is  a  production  of  bubbles,  owing  to 
fermentation  brought  about  by  the  growth  of  the  organs. 


114  BACTERIOLOGY. 

A  most  striking  form  of  development  on  potato  is 
that  possessed  by  the  organism  of  typhoid  fever  aud  the 
bacillus  of  diphtheria.  After  the  inoculation  of  a  potato 
with  either  of  these  organisms  there  is  no  naked-eye 
evidence  of  a  growth  in  either  instance,  though  micro- 
scopic examination  of  scrapings  from  the  surface  of  the 
potato  reveals  an  active  multiplication  of  the  organisms 
which  had  been  planted  there.  The  potato  is  one  of 
the  most  important  differential  media  which  we  possess 
for  this  work. 

REACTION  PRODUCED  BY  ORGANISMS  IN  THEIR 
GROWTH. — The  reactions  produced  in  the  media  by 
different  organisms  in  the  course  of  their  growth  are 
very  valuable  as  means  of  differentiation. 

In  some  cases  these  changes  are  so  marked  that  they 
are  readily  detected  by  the  coarser  reagents ;  again  they 
are  so  slight  as  to  require  the  employment  of  the  most 
delicate  indicators.  They  are  sometimes  seen  to  produce 
at  one  period  of  their  growth  an  alkaline,  at  another 
period  an  acid  reaction.  This  is  seen  in  the  cultures  of 
the  bacillus  diphtheria  of  Lotfler. 

These  differences  are  best  seen  after  the  addition  to  the 
media  in  which  the  organisms  are  to  grow  of  some  of 
the  chemical  substances  which  do  not  interfere  with  the 
development  of  the  organisms,  but  which  under  one 
reaction  are  of  one  color,  and  with  an  alteration  of  the 
reaction  become  a  different  color,  the  change  being  in- 
dicated by  the  play  of  colors.  Such  substances  as 
litmus,  in  the  form  of  tincture,  and  coralline  (rosolic 
acid)  in  alcoholic  solution  have  been  employed  for  this 
purpose.  They  may  be  added  to  the  media  in  the  pro- 
portions given  in  the  chapter  on  media,  and  the  altera- 
tions in  their  colors  studied  with  different  bacteria. 


ANILINE    DYES.  115 

Milk  and  litmus  tincture  or  peptone  solution  to  which 
rosolic  acid  has  been  added  are  very  favorable  media  for 
this  experiment. 

In  milk,  coagula  will  now  and  then  appear  as  a  result 
of  acids  produced  during  the  bacterial  life,  while  again 
acids  may  be  produced  and  yet  no  coagulation  be  noticed. 

ANILINE  DYES  FOR  DIFFERENTIAL  DIAGNOSIS. — 
The  addition  to  solid  media  of  some  of  the  aniline 
dyes,  fuchsin,  methylene-blue,  methylene-green,  and 
several  others,  as  well  as  cornbi nations  of  these  dyes, 
have  been  recommended  as  a  means  of  differentiation  of 
organisms,  the  differences  consisting  in  alterations  in  the 
color  of  the  media  due  to  oxidizing  or  reducing  proper- 
ties of  the  growing  bacteria.  As  yet  but  little  has  come 
from  this  method  of  work.  It  cannot  at  present  be 
recommended  as  a  reliable  means  of  diagnosis. 

BEHAVIOR  TOWARD  STAINING  REAGENTS. — The 
behavior  of  certain  organisms  toward  the  different  dyes 
and  their  reactions  under  special  methods  of  after-treat- 
ment serve  as  aids  to  their  diagnosis.  With  very  few 
exceptions  all  bacteria  stain  readily  with  the  common 
aniline  dyes,  but  they  differ  materially  in  the  tenacity 
with  which  they  retain  these  colors  under  the  subse- 
quent treatment  with  decolorizing  agents. 

The  tubercle  bacillus  and  the  bacillus  of  leprosy,  for 
example,  are  difficult  to  stain,  but  when  once  stained 
retain  their  color  under  the  action  of  such  energetic  de- 
colorizing agents  as  alcohol,  nitric  acid,  oxalic  acid,  etc. 

Certain  other  organisms  when  stained  with  a  solution 
of  gentian  violet  in  aniline- water,  retain  their  color  when 
treated  with  such  decolorizing  bodies  as  iodine  solution 
and  alcohol  (Gram's  method),  while  again  others  are 
completely  decolorized  by  this  method. 


116  BACTERIOLOGY. 

Many  of  them  can  only  be  treated  with  water,  or  but 
for  a  few  seconds  with  alcohol,  without  losing  their 
color. 

It  is  essential  that  these  peculiarities  should  be  care- 
fully noted  in  studying  an  organism. 

FERMENTATION. — The  production  of  gas  as  an  indica- 
tion of  fermentation  is  an  accompaniment  of  the  growtli 
of  some  organisms.  This  is  best  studied  in  media  to 
which  1  to  2  per  cent,  of  grape  sugar  has  been  added. 

In  this  experiment  the  test-tube  should  be  filled  to 
about  one-half  its  volume  with  agar-agar.  The  me- 
dium is  then  liquefied,  and  when  at  the  proper  tem- 
perature, a  small  quantity  of  a  pure  culture  of  the 
organism  under  consideration  should  be  carefully  dis- 
tributed through  it.  The  tube  is  then  placed  into  ice- 
water  and  rapidly  solidified  in  the  vertical  position. 
When  solid  it  is  placed  in  the  incubator.  After  twenty- 
four  to  thirty-six  hours,  if  the  organism  possesses  the 
property  of  causing  fermentation  of  sugar,  the  medium 
will  be  dotted  everywhere  with  very  small  cavities  con- 
taining the  gas  that  has  resulted. 

Where  it  is  important  that  the  nature  of  the  gas  thus 
produced  should  be  studied,  it  must  be  collected,  and  a 
special  form  of  apparatus  is  employed.  The  cultivation 
is  now  to  be  conducted  in  a  fluid  medium.  The  fermen- 
tation flasks  of  somewhat  the  pattern  of  that  used  by 
Einhorn  in  the  fermentation  test  for  sugar  in  the  urine 
serve  very  well  for  this  purpose. 

CULTIVATION  WITHOUT  OXYGEN. — As  we  have 
already  learned,  there  is  a  group  of  organisms  to  which 
the  name  u anaerobic  organisms"  has  been  given,  which 
are  characterized  by  their  inability  to  grow  in  the  pres- 
ence of  oxygen.  For  the  cultivation  of  the  members  oi 


CULTIVATION    WITHOUT    OXYGEN.         117 

this  group  a  number  of  devices  are  employed  for  the 
exclusion  of  oxygen  from  the  cultures. 

Koch's  method.  Koch  covered  the  surface  of  a  gelatin 
plate,  which  had  been  previously  inoculated,  with  a  thin 
sheet  of  sterilized  isinglass.  The  organisms  which  grew 
beneath  it  were  supposed  to  grow  without  oxygen. 

Hesse's  method.  Hesse  poured  sterilized  oil  upon  the 
surface  of  a  culture  made  by  stabbing  into  a  tube  of 
gelatin.  The  growth  that  occurred  along  the  track  of 
the  needle  was  supposed  to  be  anaerobic  in  nature. 

Method  of  Euchner.  The  plan  suggested  by  Buchner 
of  allowing  the  cultures  to  develop  in  an  atmosphere 
robbed  of  its  oxygen  by  pyrogallic  acid  gives  very  good 
results.  In  this  method  the  culture,  which  is  either  a 
slant  or  stab  culture  in  a  test-tube,  is  placed — tube, 
cotton  plug,  and  all — into  a  larger  tube  in  the  bottom 
of  which  has  been  deposited  1  gramme  of  pyrogallic 
acid  and  10  c.c.  of  ^  normal1  caustic  potash  solution. 
The  larger  tube  is  then  tightly  plugged  with  a  rubber 

1  A  normal  solution  is  one  that  contains  in  a  litre  as  many 
grammrnes  of  the  dissolved  substance  as  are  indicated  by  its  molec- 
ular equivalent.  The  equivalent  is  that  amount  of  a  chemical  com- 
pound which  possesses  the  same  chemical  value  as  does  one  atom  of 
hydrogen.  -For  example :  One  molecule  of  hydrochloric  acid  (HC1) 
.  has  a  molecular  weight  and  also  an  equivalent  weight  of  36.5 ;  a 
molecule  of  this  acid  has  the  same  chemical  value  as  one  atom  of 
hydrogen.  Its  normal  solution  is  therefore  36.5  grammes  to  the 
litre.  On  the  other  hand,  sulphuric  acid  (H2SO4)  contains  in  each 
molecule  two  replaceable  hydrogen  atoms ;  its  normal  solution  is 
not,  therefore,  80  grammes  (its  molecular  weight)  to  the  litre,  but 
that  amount  wrhich  would  be  equivalent  chemically  to  one  hydrogen 
atom,  viz.,  40  grammes  (one-half  its  molecular  weight)  to  the  litre. 
Abnormal  solution  of  caustic  potash  contains  as  many  grammes  to 
the  litre  as  the  number  of  its  molecular  weight — 56.1  grammes  to 
the  litre  of  water. 

6* 


118  BACTEKIOLOGY. 

stopper.  The  oxygen  is  quickly  absorbed  by  the  pyro- 
gallic  acid,  aud  the  organisms  develop  in  the  remaining 
constituents  of  the  atmosphere — nitrogen,  a  small  amount 
of  CO2,  and  a  trace  of  ammonia. 

Method  of  C.  Frdnkel.  Carl  Fraukel  suggests  the 
following  method  :  The  tube  is  first  prepared  as  if 
for  an  ordinary  Esmarch  tube.  The  cotton  plug  is 
then  replaced  by  a  rubber  stopper,  through  which 
pass  two  glass  tubes.  These  have  all  been  sterilized 
in  the  steam  sterilizer  before  using.  On  the  outer 
side  of  the  stopper  these  two  tubes  are  bent  at  right 
angles  to  the  long  axis  of  the  test-tube  into  which  they 
are  to  be  placed,  and  both  are  slightly  drawn  out  in  the 
gas  flame.  At  the  outer  extremity  of  both  of  these  tubes 
a  plug  of  cotton  is  placed ;  this  is  to  prevent  access  of 
foreign  organisms  during  manipulation.  At  the  inner 
side  of  the  rubber  stopper — that  is,  the  end  which  is  to 
be  inserted  into  the  test-tube — the  glass  tubes  are  of  dif- 
ferent lengths  :  one  reaches  to  within  0.5  cm.  of  the 
bottom  of  the  test-tube,  the  other  is  cut  off  flush  with  the 
under  surface  of  the  stopper.  This  rubber  stopper,  with 
its  glass  tubes,  is  to  replace  the  cotton  plug  of  the  test- 
tube;  the  outer  end  of  the  longer  glass  tube  is  then 
connected  with  a  hydrogen  generator  and  hydrogen 
is  allowed  to  bubble  through  the  gelatin  in  the  tube 
until  all  contained  air  has  been  expelled  and  its  place 
taken  by  the  hydrogen.  The  organisms  are  to  grow, 
then,  in  an  atmosphere  of  hydrogen.  When  all  air 
has  been  expelled,  the  two  external  ends  of  the  glass 
tubes  are  to  be  sealed  in  the  gas-flame  at  the  portions 
where  they  have  been  drawn  out.  In  sealing  the  tubes 
in  the  gas-flame  one  must  be  sure  that  all  air  has  been 

O 

expelled,  otherwise  an  explosion  is  inevitable.      This 


ESMARCH'S  METHOD.  119 

may  be  accomplished  by  allowing  the  hydrogen  to 
bubble  through  the  gelatin  for  about  ten  minutes.  The 
rubber  stopper  is  then  painted  around  with  melted 
paraffin  and  the  tube  rolled  in  the  way  given  for  ordi- 
nary Esmarch  tubes. 

During  the  operation  the  tube  containing  the  liquefied 
gelatin  should  be  kept  in  a  water-bath  of  a  temperature 
which  will  prevent  its  solidifying  and  at  the  same  time 
not  kill  the  organisms  with  which  it  has  been  inoculated. 

Method  of  Liborius.  Another  method,  that  of  Libo- 
rius,  is  to  fill  a  test-tube  about  three-quarters  full  of 
gelatin.  This  is  kept  at  the  temperature  of  boiling  water 
for  ten  minutes  to  expel  all  air  from  it.  It  is  then  to  be 
rapidly  cooled  in  ice-water,  and,  when  between  30°  C. 
and  40°  C.,  is  to  be  inoculated,  and  the  gelatin  rapidly 
solidified.  It  is  then  to  be  sealed  up  in  the  flame. 

Method  of  Kitasato  and  Weil.  For  favoring  the 
anaerobic  conditions,  Kitasato  and  Weil  have  suggested 
the  addition  to  the  culture  media  of  some  strong  re- 
ducing agent.  They  recommend  formic  acid  in  0.3  to 
0.5  per  cent. 

Esmarch 's  method.  Esmarch's  plan  is  to  prepare  in 
the  usual  way  an  Esmarch  tube  of  the  organisms,  and 
this  is  to  be  subjected  to  a  low  temperature,  and  while 
quite  cold  is  filled  with  liquefied  gelatin,  and  the  whole 
allowed  to  solidify  rapidly.  In  this  method  the  colonies 
develop  along  the  sides  of  the  tubes,  and  can  more  easily 
be  studied  than  where  they  are  mixed  through  the  gela- 
tin, as  in  the  method  of  Liborius. 

By  some  workers  the  oxygen  is  removed  by  actual 
pumping  with  the  air-pump. 

Many  other  methods  exist  for  this  special  purpose,  but 
for  the  beginner  those  given  will  suffice. 


120  BACTERIOLOGY. 

INDOL  PRODUCTION. — The  production  of  products 
other  than  those  which  give  rise  to  alterations  in  the 
reaction  of  the  media,  and  whose  presence  may  be  de- 
tected by  simple  chemical  reactions,  is  now  a  recognized 
step  in  the  identification  of  different  species  of  bacteria. 
Among  these  chemical  products  there  is  one  which  is 
produced  by  a  number  of  organisms,  and  whose  presence 
may  easily  be  detected  by  its  characteristic  behavior 
when  treated  with  certain  substances. 

Indol,  when  acted  upon  by  reducing  agents,  is  seen 
to  become  of  a  more  or  less  conspicuous  rose  color.  This 
body  was  recognized  some  time  ago  as  one  of  the  pro- 
ducts of  growth  of  the  comma  bacillus  of  Asiatic  cholera, 
and  for  a  time  was  thought  to  characterize  the  changes 
produced  in  the  media  through  the  growth  of  this  organ- 
ism. It  has  since  been  found  that  there  exist  other 
bacteria  which  also  possess  the  property  of  producing 
this  body  in  the  course  of  their  development. 

The  method  employed  for  its  detection  is  as  follows : 
Cultivate  the  organism  for  twenty-four  to  forty-eight 
hours  at  a  temperature  of  37°  C.,  in  the  simple  peptone 
solution  known  as  " Dunham's  solution"  (see  formula 
for  this  medium).  This  solution  is  preferred  because 
its  pale  color  does  not  mask  the  rose  color  of  the  reac- 
tion when  the  amount  of  indol  present  is  very  small. 

Four  tubes  should  always  be  inoculated  and  kept 
under  exactly  the  same  conditions  for  the  same  length  of 
time. 

At  the  end  of  twenty-four  or  forty-eight  hours  the 
test  may  be  made.  Proceed  as  follows  :  To  a  tube  con- 
taining 7  c.c.  of  the  peptone  solution,  but  which  has  not 
been  inoculated,  add  10  drops  of  concentrated  sulphuric 
acid.  To  another  similar  tube  add  1  c.c.  of  a  0.01  per 


INDOL    PRODUCTION.  121 

cent,  solution  of  sodium  nitrite,  and  afterward  10  drops 
of  concentrated  sulphuric  acid.  Observe  the  tubes  for 
five  to  ten  minutes.  No  alteration  in  their  color  appears, 
or  at  least  there  will  be  no  production  of  a  rose  color. 
They  contain  no  indol. 

Treat  in  the  same  way,  with  the  acid  alone,  two  of 
the  tubes  which  have  been  inoculated.  If  no  rose  color 
appears  after  five  or  ten  minutes,  add  1  c.c.  of  the  sodium 
nitrite  solution.  If  now  no  rose  color  is  produced,  the 
indol  reaction  may  be  considered  as  negative.  No  indol 
is  present. 

If  indol  is  present,  and  the  rose  color  •  appears  after 
the  addition  of  the  acid  alone,  it  is  plain  that  not  only 
indol  has  been  formed,  but  likewise  a  reducing  body. 
This  is  found,  by  proper  means,  to  be  nitrous  acid. 
The  sulphuric  acid  liberates  it  from  its  salts  and  permits 
of  its  reducing  action  being  seen. 

If  the  rose  color  appears  only  after  the  addition  of 
both  the  acid  and  the  nitrite  solution,  then  indol  has 
been  formed  during  the  growth  of  the  organisms,  but  no 
nitrites. 

Control  the  results  obtained  by  treating  the  two  re- 
maining cultures  in  the  same  way. 


CHAPTER    XII. 

Methods  of  staining — Solutions  employed — Preparation  and  stain- 
ing of  cover-slips— Preparation  of  tissues  for  section-cutting— Stain- 
ing of  tissues — Special  staining  methods. 

THE  entire  list  of  solutions  and  methods  which  huv< 
been  recommended  for  the  staining  of  bacteria  are  not 
essential  to  the  work  of  the  beginner,  so  that  only  those 
methods  which  are  of  most  common  application  will 
be  given  in  this  book.  In  general,  it  suffices  to  say, 
bacteria  stain  best  with  watery  solutions  of  the  basic 
aniline  dyes,  and  of  these,  fuchsin,  gentian-violet,  and 
methylene-blue  are  those  most  frequently  employed. 

In  practical  work  bacteria  require  to  be  stained  in 
two  conditions :  either  dried  upou  cover-slips  and  then 
stained,  or  stained  in  sections  of  tissues  in  which  they 
have  been  deposited  during  the  course  of  disease.  In 
both  processes  the  essential  point  to  be  borne  in  mind  is 
that  the  bacteria,  because  of  their  microscopic  dimen- 
sions, require  to  be  more  conspicuously  stained  than 
the  surrounding  materials  upon  the  cover-slips  or  in 
the  sections,  otherwise  their  diiferentiatiou  is  a  matter 
of  the  greatest  difficulty,  if  not  of  impossibility.  For 
this  reason,  especially  in  the  case  of  section  staining,  it 
frequently  becomes  necessary  to  decolorize  the  tissues 
after  removing  them  from  the  staining  solutions  in 
order  to  render  the  bacteria  more  prominent;  and  for 
this  purpose  special  methods,  which  provide  for  decolor- 
izatiou  of  the  tissues  without  robbing  the  bacteria  of 


COVEK-SLIP    PREPARATIONS.  123 

their  color,  are  employed.  The  ordinary  method  of 
cover-slip  examination  of  bacteria,  which  is  constantly 
in  use  in  these  studies,  is  performed  in  the  following 
way  : 

COVER-SLIP  PREPARATIONS. — In  order  that  the  dis- 
tribution of  the  organisms  upon  the  cover-slips  may  be 
uniform  and  in  as  thin  a  layer  as  possible,  it  is  essential 
that  the  slip  should  be  clean  and  free  from  grease.  For 
cleansing  the  slips  several  methods  may  be  employed. 

The  simplest  plan  with  new  cover-slips  is  to  im- 
merse them  for  a  few  hours  in  strong  nitric  acid,  after 
which  they  are  rinsed  in  water,  then  in  alcohol,  ether, 
and,  finally,  they  may  be  kept  in  alcohol  to  which  a 
little  ammonia  has  been  added.  When  they  are  to  be 
used  they  should  be  wiped  dry  with  a  clean  cotton  or 
silk  handkerchief. 

If  the  slips  have  been  previously  used,  boiling  in 
strong  soap  solution,  followed  by  rinsing  in  clean  Avarm 
water,  then  treated  as  above,  renders  them  clean  enough 
for  ordinary  purposes. 

A  method  commonly  employed  is  to  remove  all 
coarse  adherent  matter  from  slips  and  slides  by  allowing 
them  to  remain  for  a  time  in  strong  nitric  or  sulphuric 
acid.  They  are  removed  from  the  acid  after  several 
days,  rinsed  off  in  water,  and  treated  as  above.  Knauer 
has  recently  suggested  the  boiling  of  soiled  cover-slips 
and  slides  for  from  twenty  to  thirty  minutes  in  a  10  per 
cent,  watery  solution  of  lysol,  after  which  they  are  to  be 
carefully  rinsed  in  water  until  all  trace  of  the  lysol  has 
disappeared.  They  are  then  to  be  wiped  dry  with  a 
clean  handkerchief. 

Loffler's  method,  which  provides  for  the  complete 
removal  of  all  grease,  is  to  warm  the  cover-slips  in  con- 


124  BACTERIOLOGY. 

centrated  sulphuric  acid  for  a  time,  then  rinse  them 
in  water,  after  which  they  are  kept  in  a  mixture  of 
equal  parts  of  alcohol  and  ammonia.  They  are  to  be 
dried  on  a  cloth  from  which  the  fat  has  been  extracted. 
Steps  in  making  the  preparations.  Place  upon  the 
centre  of  one  of  the  clean,  dry  cover-slips  a  very  small 
drop  of  distilled  water  or  physiological  salt  solution. 
With  a  platinum  needle,  which  has  been  sterilized  in 
the  gas-flame  just  before  using  and  allowed  to  cool,  take 
up  a  very  small  portion  of  the  colony  to  be  examined 
and  mix  it  carefully  with  the  drop  on  the  slip  until 
there  exists  a  very  thin  homogeneous  film  over  the 
larger  part  of  the  surface.  This  is  to  be  dried  upon 
the  slip  by  either  allowing  it  to  remain  upon  the  table 
in  the  horizontal  position  under  a  cover,  to  protect  it 
from  dust,  or  by  holding  it  between  the  fingers  (not 
with  the  forceps),  at  some  distance  above  the  gas-flame 
until  it  is  quite  dry.  If  held  with  the  forceps  over 
the  flame  at  this  stage,  too  much  heat  may  be  uncon- 
sciously applied,  and  the  morphology  of  the  organisms  in 
the  preparation  distorted.  When  held  between  the  fin- 
gers with  the  layer  of  bacteria  away  from  the  flame  no 
such  accident  is  likely  to  occur.  When  the  whole 
pellicle  is  completely  dried  the  slip  is  to  be  taken 
up  with  the  forceps,  and,  holding  the  side  upon  which 
the  bacteria  are  deposited  away  from  the  direct  action 
of  the  flame,  is  to  be  passed  through  the  flame  three 
times,  a  little  more  than  one  second  being  allowed 
for  each  transit.  Unless  the  preliminary  drying  at  the 
low  temperature  has  been  complete,  the  preparation  will 
be  rendered  worthless  by  the  subsequent  "  fixing "  at 
the  higher  temperature,  for  the  reason  that  the  proto- 
plasm of  bacteria  when  moist  coagulates  at  these  tern- 


COVER-SLIP    PREPARATIONS.  125 

peratures,  and  in  doing  so  the  normal  outline  of  the 
cells  is  altered.  If  carefully  dried  before  fixing,  this 
does  not  occur  and  the  morphology  of  the  organism 
remains  unchanged.  A  better  plan  for  the  process  of 
fixing  is  to  employ  a  copper  plate  of  about  35  cm.  long 
by  10  cm.  wide  by  0.3  cm.  thick.  This  plate  is  laid 
upon  an  iron  tripod  and  a  small  gas-flame  is  placed 
beneath  one  of  its  extremities.  By  this  arrangement 
one  can  get  a  graduated  temperature,  beginning  at  the 
point  of  the  plate  above  the  gas-flame  where  it  is  hot- 
test, and  becoming  gradually  cooler  toward  the  other 
end  of  the  plate,  which  may  be  of  a  very  low  tempera- 
ture. By  dropping  water  upon  the  plate,  beginning  at 
the  hottest  point  and  proceeding  toward  the  cooler  end, 
it  is  easy  to  determine  the  point  at  which  the  water  just 
boils ;  it  is  at  a  little  below  this  point  that  the  cover- 
slips  are  to  be  placed,  bacteria  side  up,  and  allowed  to 
remain  about  ten  minutes,  when  the  fixing  will  be 
complete.  The  same  may  be  accomplished  in  a  small 
copper  drying-oven,  which  is  regulated  to  remain  at 
the  temperature  of  95°  to  98°  C.  This  plan  is  to 
be  preferred  to  the  process  of  passing  the  cover-slips 
through  the  flame,  as  the  organisms  are  always  subjected 
to  the  same  degree  of  heat,  and  the  distortions  which 
sometimes  occur  from  the  too  great  and  irregular  appli- 
cation of  high  temperatures  may  in  part  be  eliminated. 
The  fixing  consists  in  drying  or  coagulating  the  gelatinous 
envelope  surrounding  the  organisms,  by  which  means 
they  are  caused  to  adhere  to  the  surface  of  the  cover-slip. 
When  fixed,  the  staining  is  usually  a  simple  matter. 
The  majority  of  bacteria  with  which  the  beginner  will 
have  to  deal  stain  readily  with  solutions  of  any  of  the 
basic  aniline  dyes. 


126  BACTERIOLOGY. 

To  stain  the  fixed  preparation  it  is  taken  between  the 
forceps,  and  a  few  drops  of  a  watery  solution  of  fuchsin, 
gentian- violet,  or  metliylene-blue  are  placed  upon  the 
film  and  are  allowed  to  remain  there  twenty  to  thirty 
seconds.  The  slip  is  then  carefully  rinsed  in  water,  and 
without  drying  is  placed  bacteria  down  upon  a  slide,  the 
excess  of  water  is  taken  up  with  blotting-paper,  and  the 
preparation  is  ready  for  examination. 

Another  plan  that  is  sometimes  used  is  to  bring  the 
slip  upon  the  slide,  bacteria  clown,  without  rinsing  off 
the  staining  fluid ;  the  excess  of  fluid  is  removed  with 
blotting-paper  and  the  preparation  is  ready  for  examina- 
tion with  the  microscope.  This  method  is  satisfactory 
and  time-saving,  but  must  always  be  practised  with 
care.  The  staining  fluid  should  always  be  carefully 
filtered  before  using,  to  rid  it  of  insoluble  particles 
which  might  mislead  the  examiner  into  mistaking  them 
for  bacteria.  If  upon  examination  the  preparation 
proves  to  be  of  particular  interest,  so  that  it  is  desirable 
to  preserve  it,  then  it  is  to  be  mounted  permanently.  The 
drop  of  immersion  oil  is  to  be  removed  from  the  surface 
of  the  slip  with  blotting-paper,  and  the  slip  loosened 
from  the  slide  by  allowing  water  to  flow  around  its 
edges.  It  is  then  taken  up  with  the  forceps,  carefully 
deprived  of  the  water  adhering  to  it  by  means  of  blot- 
ting-paper, and  then  allowed  to  dry.  When  dry  it  is 
mounted  in  xylol-Cauada  balsam  by  placing  a  small 
drop  of  the  balsam  upon  the  surface  of  the  film,  and 
then  inverting  the  slip  upon  a  clean  glass  slide. 

IMPRESSION  COVER-SLIP  PREPARATIONS. — The  im- 
pression preparations  differ  in  value  from  the  ordinary 
cover-slip  preparations  only  in  one  respect :  they  present 
an  impression  of  the  organisms  as  they  were  arranged  in 


THE    ORDINARY    STAINING    SOLUTIONS.       127 

the  colony  from  which  the  preparation  was  made.  They 
are  made  by  gently  covering  the  colony  with  a  thin, 
clean  cover-slip,  lightly  pressing  upon  it,  and,  without 
moving  the  slip  laterally,  lifting  it  up  by  one  of  its 
edges.  The  organisms  adhere  to  the  slip  in  the  same 
relation  to  one  another  that  they  had  in  the  colony. 
The  subsequent  steps  of  drying,  fixing,  staining,  and 
mounting  are  the  same  as  those  just  given  for  the 
ordinary  cover-slip  preparations. 

By  this  method,  constancies  in  the  arrangement  and 
grouping  of  the  individuals  in  a  colony  can  often 
be  made  out.  Some  will  always  appear  irregularly 
massed  together,  others  will  grow  in  parallel  bundles, 
while  others,  again,  will  be  seen  as  long  twisted  threads. 

THE  ORDINARY  STAINING  SOLUTIONS. — The  solu- 
tions commonly  employed  in  staining  cover-slip  prepara- 
tions are,  as  has  been  stated,  watery  solutions  of  the  basic 
aniline  dyes  —  fuchsiu,  gentian -violet,  and  methylene- 
blue.  These  solutions  may  be  prepared  either  by  directly 
dissolving  the  dyes  in  substance  in  water  until  the  proper 
degree  of  concentration  has  been  reached,  or  by  prepar- 
ing them  from  concentrated  watery  or  alcoholic  solutions 
of  the  dyes  which  may  be  kept  on  hand  as  stock.  The 
latter  method  is  that  commonly  practised. 

The  solutions  of  the  colors  which  are  in  constant  use 
in  staining  are  prepared  as  follows  : 

Prepare  as  stock,  saturated  alcoholic  or  watery  solutions 
of  fuchsin,  gentian- violet,  and  methylene-blue.  These 
solutions  are  best  prepared  by  pouring  into  clean  bottles  , 
enough  of  the  dyes  in  substance  to  fill  them  to  about 
one- fourth  their  capacity.  The  bottle  should  then  be 
filled  with  alcohol  or  with  water,  tightly  corked,  well 
shaken,  and  allowed  to  stand  for  twenty-four  hours.  If 


128  BACTERIOLOGY. 

at  the  end  of  this  time  all  the  staining  material  has 
been  dissolved,  more  should  be  added,  the  bottle  being 
again  shaken,  and  allowed  to  stand  for  another  twenty- 
four  hours ;  this  must  be  repeated  until  a  permanent 
sediment  of  undissolved  coloring  matter  is  seen  upon  the 
bottom  of  the  bottle.  This  will  then  be  labelled  satu- 
rated alcoholic  or  watery  solution  of  fuchsin,  gentian- 
violet,  or  methylene-blue,  as  the  case  may  be.  The 
alcoholic  solutions  will  not  answer  for  staining  purposes. 

The  solutions  with  which  the  staining  is  accomplished 
are  made  from  these  alcoholic  solutions  in  the  following 
way : 

An  ordinary  test-tube  of  about  13  mm.  diameter  is 
three-fourths  filled  with  distilled  water  and  the  concen- 
trated alcoholic  or  watery  solution  of  the  dye  is  then 

FIG.  21. 


added,  little  by  little,  until  one  can  just  see  through 
the  solution.  It  is  then  ready  for  use.  Care  must  be 
given  that  the  color  does  not  become  too  dense.  The 
.best  results  are  obtained  when  it  is  just  transparent 
as  viewed  through  a  layer  of  about  12  to  14  mm. 
thick. 

These  represent  the  staining  solutions  in  everyday 
use.     They  are  kept  in  bottles  supplied  with  stoppers 


SPECIAL    STAINING    SOLUTIONS.  129 

and  pipettes  (Fig.  21),  and  when  used  are  dropped  upon 
the  preparation  to  be  stained.  After  remaining  upon 
the  preparation  for  about  thirty  seconds,  they  are 
washed  off  in  water  and  the  preparation  can  then  be 
examined. 

For  certain  bacteria  which  staiu  only  imperfectly  with 
these  simple  solutions  it  is  necessary  to  employ  some 
agent  that  will  increase  the  penetrating  action  of  the 
dyes.  Experience  has  taught  us  that  this  can  be  accom- 
plished by  the  addition  to  the  solutions  of  small  quan- 
tities of  alkaline  substances  or  by  dissolving  the  staining 
materials  in  strong  watery  solutions  of  either  aniline  oil 
or  carbolic  acid,  instead  of  simple  water. 

Of  the  solutions  thus  prepared  which  may  always  be 
employed  upon  bacteria  which  show  a  tendency  to  stain 
imperfectly,  there  are  three  in  common  use — Loffler's 
alkaline  methylene-blue  solution,  the  Koch-Ehrlich  ani- 
line-water solution  of  either  fuchsin,  gentian-violet,  or 
methyleue-blue,  and  ZiehPs  solution  of  fuchsin  in  car- 
bolic acid.  These  solutions  are  as  follows: 

Loffler's  alkaline  methylene-blue  solution : 

Concentrated  alcoholic  solution  of  methylene-blue  30  c.c. 
Caustic  potash  in  1 : 10,000  solution     .         .  100  c.c. 

Koch-Ekrlich  aniline-water  solutions.  To  about  100 
cc.  of  distilled  water  aniline  oil  is  added,  drop  by  drop, 
and  the  solution  thoroughly  shaken  after  each  addition 
until  it  is  of  an  opaque  appearance.  It  is  then  filtered 
through  moistened  filter-paper  until  the  filtrate  is  per- 
fectly clear.  To  100  c.c.  of  the  clear  filtrate  add  10  c.c. 
of  absolute  alcohol  and  11  c.c.  of  the  concentrated  al- 
coholic solution  of  either  fuchsiu,  methylene-blue,  or 
gentian-violet,  preferably  fuchsin  or  gentian-violet. 


130  BACTERIOLOGY. 

ZiehUs  carbolic-fuchsin  solution  : 

Distilled  water 100  c.c. 

Carbolic  acid  (crystalline)    ...         5  grammes. 
Alcohol        .        .        .         .         .         .10  c.c. 

Fuchsin  in  substance    ....         1  gramme. 

Or  it  may  be  prepared  by  adding  to  a  5  per  cent, 
watery  solution  of  carbolic  acid  the  saturated  alcoholic 
solution  of  fuchsin  until  a  metallic  lustre  appears  on  the 
surface  of  the  fluid. 

Both  the  Koch-Ehrlich  and  the  Ziehl  solutions 
decompose  very  quickly  after  having  been  made,  so 
that  it  is  better  to  prepare  them  when  needed  in  small 
quantities  than  to  employ  old  solutions.  Solutions  older 
than  seven  to  nine  days  should  not  be  used. 

The  three  solutions  just  given  may  be  used  for  cover- 
glass  preparations  in  the  ordinary  way. 

In  some  manipulations  it  becomes  necessary  to  stain 
the  bacteria  very  intensely,  so  that  they  may  retain  their 
color  when  exposed  to  the  action  of  decolorizing  agents. 
These  are  usually  employed  for  the  purpose  of  depriv- 
ing surrounding  objects  or  tissues  of  their  color  in  order 
that  the  stained  bacteria  may  stand  out  in  greater 
contrast.  It  is  in  these  cases  that  the  staining  solu- 
tion with  which  the  bacteria  are  being  treated  is  to  be 
warmed,  and  in  some  cases  boiled,  so  as  to  further 
increase  its  penetrating  action.  When  so  treated,  cer- 
tain of  the  bacteria  will  retain  their  .color,  even  when 
exposed  to  very  strong  djecolorizers.  The  tubercle 
bacillus  is  characterized  from  all  other  bacteria,  except 
the  bacillus  of  leprosy,  by  the  tenacity  with  which  it 
retains  its  color  when  treated  in  this  way.  It  is  an 
organism  that  is  difficult  to  stain,  but  when  once  stained 
is  equally  difficult  to  rob  of  its  color. 


STAINING    THE    TUBERCLE    BACILLUS.       131 

METHOD  OF  STAINING  THE  TUBERCLE  BACILLUS. — 
Select  from  the  sputum  of  a  tuberculous  subject  oue  of 
the  small,  white,  cheesy  masses  which  it  is  seen  to  con- 
tain. Spread  this  upon  a  cover-slip  and  dry  and  fix 
it  in  the  usual  way.  The  slip  is  now  to  be  taken  by 
its  edge  with  the  forceps  and  the  film  covered  with  a 
few  drops  of  either  the  solution  of  Koch-Ehrlich  or  of 
Ziehl.  It  is  then  held  over  the  gas-flame,  at  first  some 
distance  away,  gradually  being  brought  nearer,  until 
the  fluid  begins  to  boil.  After  it  has  bubbled  up  once  or 
twice  it  is  removed  from  the  flame,  the  excess  of  stain- 
ing washed  away  in  a  stream  of  water,  and  it  is  then 
immersed  in  a  30  per  cent,  solution  of  nitric  acid  in 
water  and  allowed  to  remain  there  until  all  the  color 
has  disappeared.  In  some  cases  this  takes  longer 
than  in  others.  One  can  always  determine  if  decoloriza- 
tion  is  complete  by  washing  off  the  acid  in  a  stream  of 
water.  If  the  preparation  is  still  quite  colored  it  should 
be  again  immersed  in  the  acid ;  if  of  only  a  very  faint 
color  it  may  be  dipped  in  alcohol,  again  washed  off 
in  water,  and  may  now  be  stained  with  some  contrast 
color.  If,  for  example,  the  tubercle  bacilli  have  been 
stained  with  fuchsin,  methyleue-blue  forms  a  "good  con- 
trast stain.  In  making  the  contrast  stain  the  steps  in 
the  process  are  exactly  those  followed  in  the  ordinary 
staining  of  cover-slip  preparations  in  general :  the 
slip  containing  the  stained  tubercle  bacilli  is  rinsed 
off  carefully  in  water  and  a  few  drops  of  the  methylene- 
blue  solution  are  placed  upon  it  and  allowed  to  remain 
for  thirty  to  forty  seconds,  when  it  is  again  rinsed  in 
water  and  examined  microscopically.  For  the  purpose 
of  observing  the  difference  between  the  behavior  of  the 
tubercle  bacilli  and  the  other  organisms  present  in  the 


132  BACTERIOLOGY. 

preparation  toward  this  method  of  staining,  it  is  well 
to  examine  the  preparation  microscopically  before  the 
contrast  stain  is  made,  then  remove  it,  give  it  the  con- 
trast color,  and  examine  it  again.  It  will  be  seen 
that  before  the  contrast  color  has  been  given  to  the 
preparation  the  tubercle  bacilli  will  be  the  only 
stained  objects  to  be  made  out,  and  the  preparation 
will  appear  devoid  of  other  organisms,  but  upon  ex- 
amining it  after  it  has  received  the  contrast  color,  a 
great  many  other  organisms  will  now  appear;  these 
will  take  on  the  second  color  employed,  while  the 
tubercle  bacilli  will  retain  their  original  color.  Before 
decolorizatiou  all  organisms  in  the  preparation  were  of 
the  same  color,  but  during  the  application  of  the  decolor- 
izing solution  all  except  the  tubercle  bacilli  gave  up  their 
color.  This  characteristic,  as  said,  serves  to  differentiate 
the  tubercle  bacillus  from  all  other  organisms,  except 
the  bacillus  of  leprosy,  which  stains  in  the  same  way  as 
does  the  bacillus  of  tuberculosis.  A  number  of  different 
methods  have  been  suggested  for  the  staining  of  tuber- 
cle bacilli,  but  the  original  method  as  employed  by 
Koch  is  so  satisfactory  in  its  results  that  it  is  not 
advisable  to  substitute  others  for  it.  The  above  differs 
from  the  original  Koch-Ehrlich  method  for  the  staining 
of  tubercle  bacilli  in  sputum  only  in  the  occasional  em- 
ployment of  Ziehl's  carbolic-fuchsin  solution  and  the 
method  of  heating  the  preparation  with  the  staining 
fluid  upon  it. 

As  Nuttall  has  pointed  out,  however,  the  strong  acid 
decolorizer  used  in  this  method  can  be  with  advantage 
replaced  by  much  more  dilute  solutions,  as  a  certain 
number  of  the  bacilli  are  entirely  decolorized  by  the 
too  energetic  action  of  the  strong  acids.  He  recom- 


GLACIAL    ACETIC    ACID    METHOD.  133 

mends  the  following  method  of  decolorization  :  After 
staining  the  slip  or  section  in  the  usual  way,  pass  it 
through  three  alcohols ;  it  is  then  to  be  washed  out  in  a 
solution  composed  of 

Water 150  c.c. 

Alcohol      .        .        .        .        .        .50  c.c. 

Concen.  sulphuric  acid       .         .         .       20  to  30  drops. 

From  this  they  are  removed  to  water  and  carefully 
rinsed.  The  remaining  steps  in  the  process  are  the 
same  as  those  given  in  the  other  methods. 

GRAM'S  METHOD. — Another  differential  method  of 
staining  which  is  very  commonly  employed  is  that  known 
as  Gram's  method.  In  this  method  the  objects  to  be 
stained  are  treated  with  an  aniline-water  solution  of 
gentian- violet  made  after  the  formula  .of  Koch-Ehrlich. 
After  remaining  in  this  for  twenty  to  thirty  minutes 
they  are  immersed  in  an  iodine  solution  composed  of 

Iodine    .         .         .         ...         .         .         1  gramme. 

Potassium  iodide  .         .         .         .         .         2  grammes. 

Distilled  water 300  c.c. 

In  this  they  remain  for  about  five  minutes  ;  they  are 
then  transferred  to  alcohol  and  thoroughly  rinsed.  If 
they  are  still  of  a  violet  color  they  are  again  treated 
with  the  iodine  solution  followed  by  alcohol,  and  this  is 
continued  until  no  trace  of  violet  color  is  visible  to  the 
naked  eye.  They  may  then  be  examined,  or  a  contrast 
color  of  carmine  or  Bismarck-brown  may  be  given  them. 

This  method  is  particularly  useful  in  demonstrating 
the  capsule  which  is  seen  to  surround  some  bacteria, 
particularly  the  diplococcus  of  pneumonia. 

GLACIAL  ACETIC  ACID  METHOD. — Another  method 
which  may  be  employed  for  demonstrating  the  presence 

7 


134  BACTERIOLOGY. 

of  the  capsule  which  surrounds  certain  organisms,  is  to 
prepare  the  cover-slips  in  the  ordinary  way,  then  cover 
the  layer  of  bacteria  upon  them  with  glacial  acetic  acid, 
which  is  instantly  poured  off  (not  washed  off  in  water), 
and  the  aniline- water  gentian-violet  solution  dropped 
upon  them ;  this  is  allowed  to  remain  three  or  four 
minutes,  is  poured  off,  and  again  a  few  drops  added, 
and  lastly  the  slip  is  washed  off  in  water.  A  very  clear, 
sharply-cut  picture  usually  follows  this  method  of  pro- 
cedure. 

STAINING  OP  SPORES. — We  have  learned  that  one  of 
the  points  by  which  spores  may  be  recognized  is  their 
refusal  to  take  up  staining  substances  when  applied  in 
the  ordinary  way.  They  may,  however,  be  stained  by 
special  methods ;  of  these  the  following  will  prove  use- 
ful :  The  cover-slip  is  to  be  prepared  from  the  material 
containing  the  spores  in  the  ordinary  way,  dried,  and 
fixed.  It  is  then  floated,  bacteria  down,  upon  the  sur- 
face of  a  watch-crystalful  of  freshly  prepared  Koch- 
Ehrlich  solution  of  fuchsiu.  This  is  then  held  by  its 
edge  with  the  forceps  about  2  cm.  above  a  very  small 
flame  of  a  Bunsen  burner,  care  being  given  that  the 
flame  touches  only  the  centre  of  the  bottom  of  the  crys- 
tal. After  a  few  seconds  the  crystal  is  elevated  gradu- 
ally until  it  is  about  6  to  8  cm.  above  the  flame,  then 
it  is  slowly  moved  down  to  the  flame  again,  and  this  up- 
and-down  movement  is  continued  until  the  staining 
fluid  begins  to  boil.  As  soon  as  a  few  bubbles  have 
been  given  off  it  is  held  aside  for  a  minute  or  two  and 
the  process  of  heating  is  repeated.  When  the  boiling 
begins,  again  the  crystal  is  held  aside  for  a  minute  or 
two.  The  crystal  is  heated  in  this  way  for  about  five 
or  six  consecutive  times.  When  the  fluid  has  stood  for 


STAINING    OF    SPORES.  135 

about  five  minutes  after  the  last  boiling,  the  preparation 
is  transferred,  without  washing  in  water,  into  a  second 
watch-crystal  containing  the  following  decolorizing  solu- 
tion : 

Absolute  alcohol 100  c.c. 

Hydrochloric  acid 3  c.c. 

In  this  solution  it  is  placed,  bacteria  up,  and  the  vessel 
is  tilted  from  side  to  side  for  about  one  minute.  It  is 
then  removed,  washed  in  water,  and  stained  with  the 
methylene-blue  solution.  The  spores  will  be  stained 
red  and  the  body  of  the  cells  will  be  blue. 

MOELLER'S  METHOD  FOR  STAINING  SPORES. — A 
method  that  has  recently  been  published  by  Moeller  is 
designed  to  favor  the  penetration  of  the  coloring  material 
through  the  spore  membrane  by  macerating  the  spores 
in  a  solution  of  chromic  acid  before  staining  them.  It 
is  as  follows : 

The  cover-slips  are  prepared  in  the  usual  way,  or  the 
fixing  may  be  accomplished  with  absolute  alcohol  instead 
of  high  temperatures.  The  preparation  is  then  held  for 
two  minutes  in  chloroform,  then  washed  off  in  water, 
then  placed  for  from  one-half  to  two  minutes  in  a  5  per 
cent,  solution  of  chromic  acid  ;  again  washed  off  in 
water,  and  now  stained  in  carbolic  fuchsin.  In  the 
process  of  staining,  the  slip  is  taken  by  the  corner  with 
the  forceps  and  carbolic  fuchsin  is  dropped  upon  the  side 
containing  the  spores.  It  is  then  held  over  the  flame 
until  it  boils,  and  then  held  some  distance  above  the 
flame  for  one  minute.  The  staining  fluid  is  then  poured 
off  and  the  preparation  is  completely  decolorized  in  5 
per  cent,  sulphuric  acid,  again  washed  off  in  water,  and 
finally  stained  for  thirty  seconds  in  the  watery  rnethy- 


136  BACTERIOLOGY. 

lene-blue  solution.  The  spores  will  be  red,  the  body  of 
the  cells  blue. 

In  this  method  the  object  of  the  preliminary  exposure 
to  chloroform  is  to  dissolve  away  any  crystals  of  lecithin, 
cholesterin,  or  fat  that  may  be  in  the  preparation,  and 
which  when  stained  might  give  rise  to  confusion. 

LOFFLER'S  METHOD  FOR  STAINING  FL.AGELL.E. — 
For  the  demonstration  of  the  locomotive  apparatus  pos- 
sessed by  motile  bacteria  we  are  indebted  to  Loftier. 
By  a  special  method  of  staining  in  which  the  use  of 
mordants  played  the  essential  part,  he  has  shown  that 
these  organisms  possess  very  delicate,  hair-like  appen- 
dages, by  the  lashing  movements  of  which  they  propel 
themselves  through  the  fluid  in  which  they  are  located. 
The  method  of  Loffler  is  as  follows : 

(1)  It  is  essential  that  the  bacteria  be  evenly  and 
not  too  numerously  distributed  upon  the  cover- slip.  The 
slips  must  therefore  be  carefully  cleansed.  (See  Laffler's 
method  of  cleaning  cover  slips.)  Five  or  six  of  the 
carefully  cleansed  cover-slips  are  to  be  placed  in  a  line 
on  the  table,  and  on  the  centre  of  each  slip  a  very  small 
drop  of  tap-water  is  placed.  From  the  culture  to  be 
examined  a  minute  portion  is  transferred  to  the  first  slip 
and  carefully  mixed  with  the  drop  of  water;  from  this 
mixture  a  small  portion  is  transferred  to  the  second, 
and  from  the  second  to  the  third  slip,  and  so  on — in  this 
way  insuring  a  dilution  of  the  number  of  organisms 
present  in  the  preparation. 

These  slips  are  then  dried  and  fixed  in  the  ordinary 
way.  They  are  next  to  be  warmed  in  the  following 
solution  : 


METHOD    FOR    STAINING    FLAGELLJ3.      137 

Tannic  acid  solution  in  water  (20  acid,  80 

water)        .......     10  c.c. 

Cold  saturated  solution  of  ferro-sulpliate       .       5  c.c. 

Saturated    watery    or    alcoholic    solution    of 

fuchsin 1  c.c. 

This  solution  represents  the  mordant.  A  few  drops 
of  it  are  to  be  placed  upon  the  film  of  bacteria  on  the 
cover-slip,  which  is  then  to  be  held  over  the  flame  until 
the  solution  begins  to  steam.  It  should  not  be  boiled. 
After  steaming,  the  mordant  is  washed  off  in  water  and 
finally  in  alcohol.  The  bacteria  are  to  be  stained  in  a 
saturated  aniline-water  fuchsin  solution. 

When  treated  in  this  way  different  bacteria  behave 
differently:  the  flagellae  of  some  stain  readily  in  the 
above  solutions ;  others  require  the  addition  of  an  alkali 
in  varying  quantities;  while  others  stain  best  after  the 
addition  of  acids.  To  meet  these  conditions  an  exact 
1  per  cent,  solution  of  caustic  soda  in  water  must  be 
prepared,  and  also  a  solution  of  sulphuric  acid  in  water 
of  such  strength  that  one  cubic  centimentre  will  be 
exactly  neutralized  by  one  cubic  centimetre  of  the 
alkaline  solution. 

For  different  bacteria  which  have  been  studied  by  this 
method,  the  one  or  the  other  of  these  solutions  is  to  be 
added  to  the  mordant  in  the  following  proportions. 

Of  the  acid  solution  : 

For  the  bacillus  of  Asiatic  cholera  .         .     5  to  1  drop. 
For  the  spirillum  rubrum        .         .         .9  drops. 

Of  the  alkaline  solution  : 

For  the  bacillus  of  typhoid  fever  .         .   1  c.c. 

For  the  bacillus  subtilis         .         .         .   28  to  30  drops. 

For  the  bacillus  of  malignant  oadema    .   36  to  37      " 


138  BACTERIOLOGY. 

For  other  organisms  one  must  determine  whether  the 
results  are  better  after  the  addition  of  acid  or  alkali, 
and  how  much  of  either  is  required.  In  general  it  may 
be  said  that  bacteria  which  produce  acids  in  the  media 
in  which  they  are  growing  require  the  addition  of  alka- 
lies to  the  mordant,  while  those  that  produce  alkalies 
require  acids  to  be  added.  By  following  Loffler's  direc- 
tions the  delicate,  hair-like  flagella3  on  motile  organisms 
may  be  rendered  plainly  visible. 

STAINING   IN    GENERAL. 

The  physics  of  staining  and  decolorization  is  hardly 
a  subject  to  be  discussed  in  a  book  of  this  character,  but, 
as  Kiihne  has  pointed  out,  solutions  which  favor  the 
production  of  diffusion  currents  facilitate  intensity  of 
staining  and  by  a  similar  process  increase  the  energy  of 
decolorizing  agents.  For  example, '  tissues  which  are 
transferred  from  water  into  watery  solutions  of  the 
coloring  matters  are  less  intensely  stained  and  more 
easily  decolorized  than  when  transferred  from  alcohol 
into  watery  staining  fluids ;  for  the  same  reason  tissues 
stained  in  watery  solutions  of  the  dyes  do  not  become 
decolorized  so  readily  when  placed  in  water  as  when 
placed  in  alcohol. 

The  diffusion  of  staining  solutions  into  the  proto- 
plasm of  dried  bacteria,  as  found  upon  cover-slip  prep- 
arations, is  much  greater  and  more  rapid  than  when 
the  same  bacteria  are  located  in  the  interstices  of  tissues. 
These  differences  are  not  in  the  bacteria  themselves, 
but  in  the  obstruction  to  diffusion  offered  by  the  tissues 
in  which  they  are  located. 

The  result  of  absence  of  diffusion  may  easily  be  illus- 


DECOLORIZING    SOLUTIONS,  139 

trated.  Prepare  a  cover-slip  preparation,  dry  it  care- 
fully, fix  it,  and  without  allowing  water  to  get  upon  it 
from  any  source,  attempt  to  stain  it  with  a  solution  of 
the  dyes  in  absolute  alcohol.  The  result  is  negative. 
The  absolute  alcohol  does  not  possess  the  property  of 
diffusing  into  the  dried  tissues,  and  hence,  as  has  been 
stated  before,  alcoholic  solutions  of  the  staining  dyes 
should  not  be  employed.  The  staining  dyes  should 
always  be  watery.1 

DECOLORIZING  SOLUTIONS. — As  regards  the  employ- 
ment of  decolorizing  agents,  it  must  always  be  borne  in 
mind  that  objects  which  are  easily  stained  are  also  easily 
decolorized,  and  those  that  can  be  caused  to  take  up  the 
staining  material  only  with  difficulty  are  also  very  diffi- 
cult to  rob  of  their  color.  The  most  common  decolorizer 
in  use  is  probably  alcohol — not  absolute  alcohol,  but 
alcohol  containing  more  or  less  of  water.  Water  alone 
has  this  property,  but  in  a  much  lower  degree  than  dilute 
alcohol.  On  the  other  hand,  a  much  more  energetic  de- 
colorization  than  that  possessed  by  either  alone  can  be 
obtained  by  alternate  exposures  to  alcohol  and  water. 
More  energetic  in  their  decolorizing  action  than  either 
water  or  alcohol,  are  solutions  of  the  acids.  They  ap- 
pear, particularly  when  they  are  alcoholic  solutions,  to 
diffuse  rapidly  into  tissues  and  bacteria  and  very  quickly 
extract  the  staining  materials  which  have  been  deposited 
there.  For  this  reason  these  solutions  should  be  em- 
ployed with  much  care. 

1  In  the  beginning  of  this  chapter  it  was  stated  that  the  saturated 
alcoholic  solutions  of  the  dyes  do  not  serve  as  stains  for  bacteria. 
It  must  be  remembered  that  this  holds  only  when  absolute  alcohol 
and  perfectly  dry  coloring  matters  have  been  used.  If  but  a  small 
proportion  of  water  is  present,  the  bacteria  may  be  stained  with  these 
solutions. 


140  BACTERIOLOGY. 

Very  dilute  acetic  acid  robs  tissues  and  bacteria  of 
their  staining  with  remarkable  activity;  still  more  ener- 
getic are  solutions  of  the  mineral  acids,  and  particularly, 
as  has  been  said,  when  this  action  is  accompanied  by  the 
decolorizing  properties  of  alcohol. 

The  acid  solutions  that  are  commonly  employed  are  : 

Acetic  acid  in  0.1  per  cent,  to  5  per  cent,  watery 
solution. 

Nitric  acid  in  20  per  cent  to  30  per  cent,  watery 
solution. 

Hydrochloric  acid  in  3  per  cent,  solution  in  alcohol. 

STAINING   OF   BACTERIA   IN   TISSUES. 

In  staining  tissues  for  the  purpose  of  demonstrating 
the  bacteria  which  they  may  contain,  a  number  of  points 
must  be  borne  in  mind :  the  conditions  which  favor  the 
diffusion  of  the  staining  fluids  into  the  bacteria -are  now 
not  so  favorable  to  a  rapid  staining  as  they  were  when 
the  bacteria  alone  were  present  upon  cover-slips ;  the 
staining  of  tissues  therefore  requires  a  longer  exposure 
to  the  dyes  than  with  the  cover-slips.  In  tissues,  too, 
there  are  other  substances  beside  the  bacteria  which 
become  stained,  and  these,  unless  robbed  in  whole  or  in 
part  of  their  color,  may  so  mask  the  stained  bacteria  as 
to  render  them  difficult,  if  not  impossible  of  detection. 
Tissues  must  therefore  always  be  subjected  to  some 
degree  of  decolorizatiou,  and  this  must  be  practised 
without  depriving  the  bacteria  of  their  color. 

The  details  of  the  methods  of  decolorization  will  be 
described  in  the  section  on  the  technique  of  staining. 

Another  point  to  be  remembered  in  staining  tissues  is 
that  they  can  never  be  heated  and  retain  their  structure, 


STAINING    OF    BACTEKIA    IN    TISSUES.      141 

in  the  same  way  that  one  heats  cover-slips.  The  best 
results  are  not  obtained  in  efforts  to  hasten  the  staining 
by  subjection  to  high  temperatures,  but  rather  by  longer 
exposures  at  lower  temperatures. 

HARDENING  THE  TISSUES. — The  bits  of  tissue — not 
greater  than  1  cm.  cube — are  to  be  placed,  as  fresh  as 
possible,  in  absolute  alcohol.  The  bit  of  tissue  should 
rest  upon  a  pad  of  cotton  or  filter-paper  in  the  bottle  con- 
taining the  alcohol,  in  order  that  it  may  be  elevated  and 
surrounded  by  the  part  of  the  alcohol  which  is  specifically 
the  lightest,  and  consequently  contains  least  water.  The 
alcohol  abstracts  water  from  the  tissue,  and,  as  the 
dehydration  proceeds,  the  tissue  becomes  accordingly 
more  and  more  dense.  When  of  about  the  consistency 
of  fresh  solid  rubber,  or  preferably  not  quite  so  dense,  it 
is  ready  to  cut.  A  small  portion  of  about  0.5  cm.  cube 
should  be  cemented  to  a  bit  of  cork  with  ordinary 
mucilage,  and  allowed  to  remain  in  the  open  air  for  a 
minute  or  two  for  the  mucilage  to  harden.  Alcohol  should 
be  dropped  upon  it  occasionally,  to  prevent  drying  of 
the  tissue.  When  the  mucilage  is  hard,  the  cork  with 
the  piece  of  tissue  upon  it  may  be  left  in  alcohol  over 
night,  and  on  the  following  day  it  may  be  cut. 

SECTION-CUTTING. — This  is  accomplished  by  the  use 
of  an  instrument  known  as  a  microtome  (Fig.  22).  It  is 
an  apparatus  provided  with  a  clamp  for  holding  the  cork 
upon  which  the  tissue  is  cemented  and  also  a  sliding 
clamp  which  carries  a  knife.  The  tissue  is  clamped 
horizontally,  and  the  knife  is  caused  to  slide  across  its 
upper  surface,  also  in  the  horizontal  direction.  Beneath 
the  clamp  for  holding  the  tissue  is  a  milled  disc,  by 
means  of  which  a  screw  is  caused  to  revolve,  and  in 
revolving  raises  or  lowers  the  clamp  holding  the  tissue, 


142 


BACTERIOLOGY. 


so  that  the  tissue  may  be  brought  closer  to  or  farther 
from  the  plane  in  which  the  knife  slides.  By  this 
arrangement  sections  of  any  desired  thickness  can  be  cut 
by  turning  the  milled  disc  with  the  one  hand  and  causing 
the  knife  to  traverse  the  tissue  with  the  other. 

The  tissue  and  the  knife-blade  should  be  kept  wet 
with  alcohol,  so  that  the  sections  may  float  upon  the 
blade  of  the  knife,  from  which  they  can  be  easily 
removed  without  tearing,  with  a  curved  needle  or  a 
camel-hair  pencil.  As  the  sections  are  cut  they  are 
placed  in  a  dish  containing  alcohol. 

FIG.  22. 


There  are  some  tissues  which, *by  reason  of  their  his- 
tological  structure,  do  not  become  sufficiently  dense  when 
exposed  to  alcohol  to  permit  of  their  being  cut  in  the 
above  way.  It  becomes  necessary  to  render  them  more 
solid  by  filling  their  interstices  with  some  substance  that 


STAINING    OF    BACTEKIA    IN    TISSUES.       143 

neither  interferes  with  their  structure  nor  prevents  their 
being  cut  into  sections.  They  must  be  "  imbedded,"  as 
this  process  is  called. 

Imbedding  in  celloidin.  Most  convenient  for  this 
purpose  is  celloidiu,  a  body  somewhat  similar  to  col- 
lodion, soluble  in  a  mixture  of  equal  parts  of  alcohol 
and  ether,  as  well  as  in  absolute  alcohol. 

Two  solutions  of  celloidin  are  to  be  employed,  the  one 
a  thin  solution  in  a  mixture  of  absolute  alcohol  and 
ether,  equal  parts,  the  other  a  thick  solution  in  absolute 
alcohol.  Into  the  thin  solution  the  tissue  is  placed  from 
absolute  alcohol,  and  allowed  to  remain  for  twenty-four 
or  forty-eight  hours.  It  is  then  placed  in  the  thick 
solution  for  one  or  two  days.  From  this  it  may  be 
removed  and  placed  immediately  upon  a  bit  of  cork. 
The  adherent  celloidin  will  act  as  a  cement,  and  as  it 
hardens  rapidly,  the  tissue  is  soon  fast  to  the  cork  ;  after 
remaining  in  60  per  cent,  alcohol  for  twenty-four  hours 
to  complete  the  solidification  of  the  celloidin,  sections 
may  be  cut  as  in  the  way  just  described  for  tissues  not 
so  treated. 

The  paraffin  method  of  imbedding  is  not  to  be  recom- 
mended for  bacteriological  purposes. 

STAINING  OF  THE  SECTIONS. — The  sections  when  cut 
may  be  stained  in  a  variety  of  ways.  The  ordinary 
watery  solutions  of  the  three  common  basic  aniline  dyes 
— fuchsin,  gentian-violet,  or  methylene-blue — or,  what 
is  better,  the  alkaline  methylene-blue  solution  of  Loffler, 
may  be  employed  for  general  use. 

The  acid  aniline  dyes,  as  well  as  some  of  the  vege- 
table coloring  matters,  are  essentially  nuclear  stains,  and 
are  not  applicable  to  the  staining  of  bacteria. 

Into  a  watch-glass  containing  either  of  the  staining 


144  BACTERIOLOGY. 

solutions  mentioned,  the  sections  are  to  be  placed  after 
having  been  in  water  for  about  one  minute.  They 
remain  in  the  staining  solutions  for  from  five  to  eight 
minutes.  They  are  then  removed,  rinsed  in  water,  and 
partly  decolorized  in  0.1  per  cent,  acetic  acid  for  only  a 
few  seconds;  again  washed  out  in  water,  then  in  abso- 
lute alcohol  for  a  few  seconds,  and  from  this  again  into 
absolute  alcohol  for  the  same  time,  and  finally  into  cedar 
oil  or  xylol.  Here  they  remain  for  from  one-half  to  three- 
fourths  of  a  minute.  They  are  now  to  be  carefully  spread 
out  upon  a  spatula,  which  is  held  in  the  fluid  under 
them,  and  without  draining  off  the  fluid  are  transferred 
to  a  clean  glass  slide.  This  must  be  done  carefully  to 
avoid  tearing  The  easiest  way  to  do  this  is  to  hold  the 
spatula  on  which  the  section  floats  in  one  hand,  with  its 
point  just  touching  the  surface  of  the  glass  slide,  and 
then  with  a  needle  pull  the  section  gently  off  upon  the 
slide.  The  fluid  comes  with  it,  and  the  floating  section 
may  be  easily  spread  out  into  a  flat  surface.  The 
excess  of  fluid  is  taken  up  with  blotting-paper,  after 
which  a  drop  of  xylol-balsam  is  placed  upon  the  centre 
of  the  section,  and  is  then  covered  with  a  thin,  clean 
cover-slip.  It  is  now  ready  for  examination. 

Each  step  in  the  above  process  has  its  definite  object. 
The  sections  are  placed  in  water  before  staining  in  order 
that  the  diffusion  of  the  staining  solution  into  the  tissues 
may  be  diminished ;  otherwise  our  efforts  at  rendering 
the  bacteria  more  conspicuous  by  decolorizing  the  tissues 
in  which  they  are  located  would  rob  the  bacteria  of  their 
color  as  well. 

The  acetic  acid  and  also  the  alcohol  are  decolorizers, 
and  are  directed  toward  the  excess  of  staining  in  the 
tissues.  The  cedar  oil  or  xylol  are  bodies  which  mix 
on  the  one  hand  with  alcohol,  on  the  other  with  balsam. 


STAINING    OF    BACTERIA    IN    TISSUES.      145 

They  are  known  as  "  clearing  fluids,"  and  fill  up  the 
gap  that  would  otherwise  be  left  in  the  process,  for  a 
section  cannot  be  mounted  in  balsam  directly  from  alco- 
hol ;  the  two  bodies  do  not  mix  perfectly. 

A  number  of  clearing  agents  are  in  general  use ;  in 
fact,  almost  all  the  essential  oils  come  under  this  head. 
There  is  one — oil  of  cloves — which  is  very  commonly 
used  in  histological  work,  but  it  must  not  be  employed 
in  tissues  containing  bacteria.  It  not  only  extracts  too 
much  color  from  the  bacteria,  but  causes  them  to  fade 
after  the  sections  have  been  mounted  for  a  time. 

When  the  section  thus  stained  and  mounted  is  ex- 
amined microscopically,  it  may  be  found  that  the  tissues 
still  possess  so  much  color  that  the  bacteria  are  not 
visible,  in  which  case  they  have  not  been  decolorized 
sufficiently ;  or,  on  the  other  hand,  both  bacteria  and 
tissues  may  have  parted  with  their  stains — then  decol- 
orization  has  been  carried  too  far.  In  either  case  the 
fault  must  be  remedied  in  the  manipulation  of  the  next 
section  to  be  mounted. 

In  short,  the  steps  in  the  process  of  staining  sections 
in  general  are  these : 

a.  From  alcohol  into  distilled  water  for  one  minute. 

6.  Into  the  staining  fluid  for  from  five  to  eight 
minutes. 

c.  Into  water  for  from  three  to  five  minutes. 

d.  Into  0.1  per  cent,  acetic  acid  for  about  one-half 
minute. 

e.  Absolute  alcohol  for  a  few  seconds. 
/.  Absolute  alcohol  for  a  few  seconds. 
g.  Xylol  for  about  one-half  minute. 

h.  Removal  with  spatula  or  section -lifter  to  slide. 
i.   Removal  of  excess  of  xylol. 
j.  Mounting  in  xylol-balsam. 


146  BACTERIOLOGY. 

The  section  must  be  lifted  from  one  vessel  to  the 
other  by  means  of  either  a  curved  needle  or  a  glass  rod 
drawn  out  to  a  fine  end  and  bent  in  the  form  of  a  curved 
needle. 

By  the  above  process  of  staining,  which  can  be  prac- 
tised as  a  general  method  for  most  bacteria  in  tissues, 
the  nuclei  of  the  tissue  cells,  as  well  as  the  bacteria,  will 
be  more  or  less  deeply  stained. 

SPECIAL  METHODS  OF  STAINING  BACTERIA  IN 
TISSUES. — For  purposes  of  contrast  stains  it  some- 
times becomes  necessary  to  completely,  or  nearly  com- 
pletely, decolorize  the  tissues  and  leave  the  bacteria 
unaltered  in  color.  For  this  purpose  special  methods 
depending  on  the  staining  peculiarities  of  the  bacteria 
under  consideration  have  been  devised. 

Gram's  method  with  tissues.  One  of  the  most  commonly 
employed  differential  stains  is  that  of  Gram.  In  gene- 
ral, it  is  practised  in  the  way  given  for  its  employment 
on  cover-slip  preparations  with  some  slight  modifications. 

In  this  method  the  sections  are  to  be  placed  from 
water  into  a  solution  of  aniline-water  gentian-violet, 
as  prepared  by  the  Koch-Ehrlich  formula,  but  which  has 
been  diluted  with  about  one-third  its  volume  of  water. 
In  this  the  sections  remain  for  about  ten  minutes,  prefer- 
ably in  a  warm  place,  at  a  temperature  of  about  40°  C. 
They  should  never,  under  any  conditions,  be  boiled. 

From  this  they  are  washed  alternately  in  the  iodine 
solution  and  alcohol,  occasionally  renewing  the  stained 
with  clean  alcohol,  until  all  color  has  been  extracted 
from  them.  They  are  then  brought  for  one  minute  into 
a  dilute  watery  solution  of  eosin  or  safranin  or  into  picro- 
carmine ;  again  washed  out  for  a  few  seconds  in  alcohol, 
and  finally  for  one-fourth  minute  in  absolute  alcohol. 


STAINING    OF    BACTERIA    IN    TISSUES.      147 

From  this  they  are  transferred  to  xylol  for  a  half- 
minute.  The  remaining  steps  in  the  process  are  the 
same  as  those  given  in  the  general  method.  In  some 
cases  better  results  are  obtained  by  reversing  the  steps 
in  the  process  and  staining  the  bacteria  last,  for  then  the 
frequent  decolorizing  action  of  the  alcohol  on  the  bacteria 
is  diminished;  thus,  place  the  sections  from  alcohol  into 
picro-carmine  for  one-half  hour,  then  wash  out  in  50 
per  cent,  alcohol,  then  for  from  three  to  five  minutes 
in  the  dilute  aniline- water  gentian- violet  solution,  then 
into  the  iodine  bath,  after  three  minutes  wash  out  in 
alcohol,  and,  finally,  for  one- fourth  minute  in  abso- 
lute alcohol,  and  then  into  the  xylol,  from  which  they 
may  be  mounted.  The  organisms  which  may  be  stained 
by  this  method  are  mic.  tetragenus,  b.  diphtherise,  b. 
anthracis,  staph.  pyogenes  aureus,  and  a  few  others. 
It  cannot  be  successfully  employed  with  the  bacillus 
of  typhoid  fever. 

Staining  with  dahlia  and  decolorizing  with  soda  solu- 
tion. Another  method  that  is  not  very  commonly  em- 
ployed, though  the  results  obtained  by  its  use  are  in 
many  cases  very  satisfactory,  is  to  stain  the  tissues  in  a 
strong  watery  solution  of  dahlia  (about  one-fourth  satu- 
rated) for  from  ten  to  fifteen  minutes;  from  this  they 
are  brought  into  a  two  per  cent,  solution  of  sodium  or 
potassium  carbonate,  and  from  this  into  alcohol,  alter- 
nating from  the  one  to  the  other,  until  the  section  is 
almost  colorless.  From  the  alcohol  they  are  rinsed  out 
in  water  and  then  brought  into  a  dilute  watery  solution 
of  either  eosin,  Bismarck-brown,  or  safranin  for  one 
minute,  then  washed  out  in  alcohol,  finally  into  absolute 
alcohol,  and  then  into  xylol,  from  which  they  may  be 
mounted  in  the  manner  given. 


148  BACTERIOLOGY. 

Especially  brilliant  results  are  obtained  when  tissues 
containing  anthrax  bacilli  are  stained  by  this  process ; 
the  bacilli  will  be  of  a  deep-blue  color,  while  the  sur- 
rounding tissues  will  be  of  the  color  used  as  contrast. 

Kiihne's  carbolic  methylene-blue  method.  Stain  the 
sections  in  the  following  solution  for  from  one-half  to 
one  hour : 

Methylene-blue,  in  substance        .        .       1.5  grammes. 
Absolute  alcohol 10  c.c. 

Rub  up  thoroughly  in  a  mortar,  and  when  the  blue 
is  completely  dissolved,  add  gradually  100  c.c.  of  a  5 
per  cent,  solution  of  carbolic  acid.  (The  solution  de- 
composes after  a  short  time ;  it  should  be  made  fresh 
when  needed.)  From  this  the  sections  are  washed  out 
in  water,  then  in  1.5  to  2  per  cent,  hydrochloric  acid  in 
water,  from  this  into  a  solution  of  lithium  carbonate  of 
the  strength  of  six  to  eight  drops  of  a  concentrated 
watery  solution  of  the  salt  to  ten  drops  of  water,  and 
from  this  again  thoroughly  washed  in  water ;  then  into 
absolute  alcohol  containing  enough  methylene-blue  in 
substance  to  give  it  a  tolerably  dense  color,  then  for 
a  few  minutes  into  aniline  oil  to  which  a  little  methy- 
lene-blue in  substance  has  been  added ;  then  com- 
pletely rinse  out  in  pure  aniline  oil,  from  this  into 
thymol  or  oil  of  turpentine  for  tsvo  minutes,  and  then 
into  xylol,  from  which  they  are  mounted  in  xylol-balsam. 
The  advantages  of  this  method  are  that  it  is  generally 
applicable,  and  by  its  use  the  bacteria  are  not  robbed  of 
their  color,  whereas  the  tissues  are  sufficiently  decolor- 
ized to  render  the  bacteria  visible  and  admit  of  the  use 
of  contrast  stains. 

Weigert's  modification  of  Gram's  method  for  sections. 
Stain  the  sections  in  Ehrlich's  aniline-  water  gentian- violet 


STAINING    OF    BACTERIA    IN    TISSUES.       149 

solution  for  five  or  six  minutes ;  wash  out  in  water  or 
physiological  salt  solution  (0.6  to  0.7  per  cent,  solution 
of  sodium  chloride  in  distilled  water) ;  transfer  them 
with  the  section-lifter  to  the  slide ;  take  up  the  excess 
of  fluid  by  gently  pressing  upon  the  flat  section  with 
blotting-paper ;  treat  the  section  with  the  iodine  solu- 
tion used  by  Gram ;  take  up  the  excess  of  the  solution 
with  blotting-paper ;  cover  the  section  with  aniline  oil — 
this  not  only  differentiates  the  component  parts  of  the 
section,  but  dehydrates  as  well ;  wash  out  the  aniline 
oil  with  xylol,  and  mount  in  the  usual  way  in  xylol- 
balsam.  Or,  decolorization  with  iodine  may  be  omitted, 
and  the  sections,  after  staining  in  the  aniline-water  gen- 
tian-violet for  five  or  six  minutes  or  longer,  if  necessary, 
are  transferred  to  the  slide  without  being  washed  in 
water  or  salt  solution,  or  if  so  only  very  slightly  and 
rapidly,  dried  as  completely  as  possible  with  filter-paper, 
then  are  decolorized  with  a  mixture  of  aniline  oil  (one 
part)  and  xylol  (two  parts).  This  is  the  delicate  part  of 
the  process  and  can  be  watched  under  the  low  power  of 
the  microscope  ;  when  decolorization  is  sufficient  (re- 
peated applications  of  the  aniline  oil  and  xylol  mixture 
are  generally  necessary),  pure  xylol  replaces  the  mixture, 
and  the  specimen  is  finally  mounted  in  xylol  balsam. 
Unless  all  the  aniline  oil  is  replaced  by  the  xylol  the 
specimen  will  not  keep  well.  In  this  process  the  aniline 
oil  is  really  the  decolorizer  and  has  the  valuable 
property  of  absorbing  a  certain  amount  of  water,  so 
that  dehydration  with  alcohol  is  avoided.  This  method, 
while  it  stains  certain  bacteria  in  tissues  very  satisfac- 
torily, is  nevertheless  designed  especially  for  the  stain- 
ing of  fibrin.  Fibrin  and  hyaline  materal  will  be 
stained  deep  blue,  bacteria  a  dark  violet. 


150  BACTERIOLOGY. 

STAINING  FOR  TUBERCLE  BACILLI  IN  TISSUES. — As 
for  the  staining  of  cover-slips,  only  those  methods  most 
commonly  employed  will  be  given. 

The  method  of  Ehrlich.  Stain  the  sections  in  aniline- 
water  fuchsia  or  gentian-violet  for  twenty-four  hours ; 
decolorize  in  20  per  cent,  nitric  acid  for  a  few  seconds 
only,  the  color  need  not  be  entirely  extracted  ;  then  into 
70  per  cent,  alcohol  until  no  more  color  can  be  ex- 
tracted by  the  alcohol ;  stain  as  contrast  color  in  dilute 
watery  methyleue-blue,  malachite-green,  or  Bismarck- 
brown  solution ;  wash  out  in  90  per  cent,  alcohol,  then 
in  absolute  alcohol  for  a  few  seconds;  clear  up  in  xylol 
and  mount  in  xylol-balsam. 

Method  of  Ziehl-Neelsen.  Stain  the  sections  in 
warmed  carbol-fuchsin  solution  for  one  hour ;  tempera- 
ture to  be  about  45°  to  50°  C.  Decolorize  for  a  few 
seconds  in  5  per  cent,  sulphuric  acid,  then  in  70  per 
cent,  alcohol,  and  from  this  on  as  by  the  Ehrlich 
method. 

Dry  method.  For  the  tubercle  bacilli,  as  for  many 
other  organisms  in  tissues,  the  following  method  may 
be  employed  if  only  the  presence  of  organisms  is  to 
be  detected  and  the  histological  condition  of  the  tissues 
is  a  matter  of  no  consequence  :  Bring  the  sections  from 
water  upon  a  slide  or  cover-slip,  dry,  fix,  and  stain  by 
the  methods  for  cover-slip  preparations. 


CHAPTEE    XIII. 

Inoculation  of  animals — Subcutaneous  inoculation,  intra-venous 
injection. 

AFTER  subjecting  an  organism  to  the  methods  of  study 
that  we  have  just  reviewed,  it  remains  that  its  action 
upon  the  lower  animals  should  be  tested — i.  e.,  to  deter- 
mine if  it  possesses  the  property  of  producing  disease  or 
not,  and  if  so,  what  are  the  pathological  results  of  its 
growth  in  the  tissues  of  these  animals,  and  in  what  way 
must  it  gain  entrance  to  the  tissues  in  order  to  produce 
these  results. 

This  is  commonly  determined  by  both  subcutaneous 
and  intra-venous  inoculation. 

SUBCUTANEOUS  INOCULATION  OF  ANIMALS. — The 
animals  usually  employed  in  the  laboratory  for  purposes 
of  inoculation  are  white  mice,  gray  house-mice,  guinea- 
pigs,  rabbits,  and  pigeons. 

For  simple  subcutaneous  inoculation  the  steps  in  the 
process  are  practically  the  same  in  all  cases.  The  hair 
or  feathers  are  to  be  carefully  removed.  If  the  skin 
is  very  dirty  it  may  be  scrubbed  with  soap  and  water. 
Disinfection  of  the  skin  is  impossible,  so  that  it  need  not 
be  attempted.  If  the  inoculation  is  to  be  by  means  of  a 
hypodermatic  syringe,  then  a  fold  of  the  skin  may  be 
lifted  up  and  the  needle  inserted  in  the  way  common 
to  this  procedure.  If  a  solid  culture  is  to  be  inocu- 
lated, a  fold  of  the  skin  may  be  taken  up  with  the 
forceps  and  a  pocket  cut  into  it  with  scissors  which  have 


152  BACTERIOLOGY. 

previously  been  sterilized.  This  pocket  must  be  cut 
large  enougli  to  admit  the  end  of  the  needle  without  its 
touching  the  sides  of  the  opening  as  it  is  inserted. 
Beneath  the  skin  will  be  found  the  superficial  and  deep 
connective-tissue  fasciae.  These  must  be  taken  up  with 
sterilized  forceps  and  with  sterilized  scissors,  and  in- 
cised in  a  way  corresponding  to  the  skin.  The  pocket 
is  then  to  be  held  open  with  the  forceps  and  the  sub- 
stance to  be  inserted  is  introduced  as  far  back  under 
the  skin  and  fasciae  as  possible,  care  being  taken  not  to 
touch  the  edges  of  the  wound  if  it  can  be  avoided. 
The  wound  may  be  then  simply  pulled  together  and 
allowed  to  remain.  No  stitching  or  efforts  at  closing 
it  are  necessary. 

During  manipulation  the  animal  must  be  held  quiet. 
For  this  purpose  special  forms  of  holders  have  been 
devised,  but  if  an  assistant  is  to  be  obtained  for  the 
operation,  the  simple  subcutaneous  inoculation  may  be 
made  without  the  aid  of  a  mechanical  holder. 

For  mice,  however,  a  holder  is  of  much  convenience. 
This  piece  of  apparatus  consists  of  a  bit  of  board  of  about 
7  x  10  cm.  and  2  cm.  thick,  upon  which  is  tacked  a  hol- 
low, tapering  roll  of  wire  gauze,  a  truncated  cone  of 
about  6  cm.  long  and  of  about  1.5  cm.  in  diameter  at 
one  end  and  2  cm.  at  its  other  end. 

This  is  tacked  upon  the  board  in  such  a  position  that 
its  long  axis  runs  in  the  long  diameter  of  the  board, 
being  equidistant  from  its  two  sides.  Its  small  end  is 
placed  at  the  edge  of  the  board.  The  mouse  is  taken  up 
by  the  tail  by  means  of  a  pair  of  tongs  and  allowed  to 
crawl  into  the  smaller  end  of  this  wire  cone.  When  so 
far  in  that  only  the  root  of  the  tail  projects,  the  animal 
is  then  fixed  in  this  position  by  a  clamp  and  thumb- 


SUBCUTANEOUS    INOCULATION.  153 

screw,  with  which  the  apparatus  (Fig.  23)  is  provided. 
The  animal  usually  remains  perfectly  quiet  and  may  be 
handled  without  difficulty. 

FIG.  23. 


The  hair  from  over  the  root  of  the  tail  is  to  be  care- 
fully cut  away  with  the  scissors  and  a  pocket  cut  through 
the  skin  at  this  point.  The  inoculation  is  then  made 
into  the  loose  tissues  under  the  skin  over  this  part  of  the 
back  in  the  same  way  that  has  just  been  described.  It 
is  best  always  to  insert  the  needle  some  distance  along 
the  spinal  column  and  thus  deposit  the  material  as  far 
from  the  surface-wound  as  possible. 

As  the  subcutaneous  operation  is  very  simple  and  takes 
only  a  few  moments,  guinea-pigs,  rabbits,  and  pigeons 
are  best  held  by  an  assistant.  The  front  legs  in  the  one 
hand  and  the  hind  legs  in  the  other,  with  the  animal 
stretched  upon  its  back  on  a  table,  is  the  usual  position 
for  the  operation  when  practised  upon  guinea-pigs  and 
rabbits.  The  point  at  which  the  inoculations  are  com- 
monly made  is  in  the  abdominal  walls  either  to  the  right 
or  left  of  the  median  line  and  about  3  cm.  distant.  When 
pigeons  are  used  they  are  held  with  the  legs,  tail,  and 
ends  of  the  wings  in  the  one  hand,  and  the  head  and 
anterior  portion  of  the  body  in  the  other,  leaving  the 


154  BACTERIOLOGY. 

area  occupied  by  the  pectoral  muscles,  over  which  the 
inoculation  is  to  be  made,  free  for  manipulation.  The 
hair  should  be  closely  cut  with  the  scissors  in  the  case 
of  the  guinea-pigs  and  rabbits,  and  the  feathers  pulled 
out  in  the  case  of  the  pigeon. 

INJECTION  INTO  THE  CIRCULATION. — It  is  not  infre- 
quently desirable  to  inject  the  material  under  considera- 
tion directly  into  the  circulation  of  an  animal.  If  the 
rabbit  is  to  be  employed  for  the  purpose,  the  operation 
is  usually  done  upon  one  of  the  veins  in  the  ear. 

To  those  who  have  had  no  practice  in  this  procedure 
it  offers  a  great  many  difficulties;  but  if  the  directions 
which  will  be  given  be  strictly  observed,  the  greatest  of 
these  obstacles  to  the  successful  performance  of  the  oper- 
ation may  be  overcome. 

When  viewing  the  circulation  in  the  ear  of  the  rabbit 
by  transmitted  light,  three  conspicuous  branches  of  the 
main  vessel  (vena  auricularis  posterior)  will  be  seen. 
One  runs  about  centrally  in  the  long  axis  of  the  ear, 
one  runs  along  its  anterior  margin,  and  one  along  its 
posterior  margin.  The  central  branch  (the  ramus  ante- 
rior of  the  vena  auricularis  posterior)  is  the  largest  and 
most  conspicuous  vessel  of  the  ear,  and  is,  therefore, 
selected  by  the  inexperienced  as  the  branch  into  which 
it  would  appear  easiest  to  insert  a  hypodermatic  needle. 
This,  however,  is  fallacious.  This  vessel  lies  very  loosely 
imbedded  in  connective  tissue,  and  in  efforts  to  introduce 
a  needle  into  it,  rolls  about  to  such  an  extent  that 
only  after  a  great  deal  of  difficulty  does  the  experiment 
succeed.  On  the  other  hand,  the  posterior  branch 
(ramus  lateralis  posterior  of  the  vena  auricularis  poste- 
rior) is  a  very  fine,  delicate  vessel  which  runs  along  the 
posterior  margin  of  the  ear,  and  which  is  so  firmly  fixed 


INJECTION    INTO    THE    CIRCULATION.       155 

in  the  dense  tissues  which  surround  it  that  it  is  prevented 
from  rolling  about  under  the  point  of  the  needle.  The 
further  away  from  the  mouth  of  the  vessel — that  is,  the 
nearer  we  approach  its  capillary  extremity — the  more 
favorable  become  the  conditions  for  the  success  of  the 
operation. 

Select,  then,  the  very  delicate  vessel  lying  quite  close 
to  the  posterior  margin  of  the  ear,  and  make  the  injec- 
tion as  near  to  the  apex  of  the  ear  as  possible.  The 
injection  is  always  to  be  made  from  the  dorsal  surface  of 
the  ear. 

Of  no  less  importance  than  the  selection  of  the  proper 
vessel,  is  the  shape  of  the  point  of  the  needle  employed. 

The  hypodermatic  needles  as  they  come  from  the 
makers  are  not  suited  at  all  for  this  operation  be- 
cause of  the  way  in  which  their  points  are  ground. 
If  one  examines  carefully  the  point  of  a  new  hypo- 
dermatic needle  it  will  be  seen  that  the  long  point,  in- 
stead of  presenting  a  flat,  slanting  surface,  when  viewed 
from  the  side,  is  more  or  less  of  a  curved  surface. 
Now,  in  efforts  to  introduce  such  a  needle  into  vessel  a 
of  very  small  calibre,  it  is  commonly  seen  that  the  ex- 
treme point  of  the  needle,  instead  of  remaining  in  the 
vessel  as  it  would  do  were  it  straight,  very  commonly 
projects  into  the  opposite  wall,  and  as  the  needle  is  in- 
serted further  and  further  into  the  tissues,  it  is  usually 
pushed  through  the  vessels  into  the  loose  tissues  beyond, 
and  the  material  to  be  injected  is  deposited  into  these 
tissues  instead  of  into  the  circulation.  If,  on  the  con- 
trary, the  slanting  point  of  the  needle  is  ground  down 
until  its  surface  is  perfectly  flat,  and  when  viewed  from 
the  side  no  more  curvature  exists,  then  when  once  in- 
serted into  a  vessel  it  usually  remains  there,  and  there 


156  BACTERIOLOGY. 

is  no  tendency  to  penetrate  through  the  opposite  wall. 
We  never  use  a  new  hypodermatic  needle  until  its  point 
is  carefully  ground  down  to  a  perfectly  flat,  slanting 
surface  and  no  more  curvature  exists. 

These  differences  may  perhaps  come  out  clearer  if 
represented  diagrammatically. 

FIG.  24. 
A 


In  Fig.  24,  A,  the  needle  has  the  point  usually  seen 
when  new. 

In  Fig.  24,  B,  the  point  has  been  ground  down  to  the 
shape  best  suited  for  this  operation. 

The  needles  need  not  be  returned  to  the  maker.  One 
can  grind  them  to  the  shape  desired  in  a  few  minutes 
upon  an  oilstone. 

The  size  of  the  needle  is  that  commonly  employed  for 
subcutaneous  injections. 

When  the  operation  is  to  be  performed,  an  assistant 
holds  the  animal  gently  but  firmly  in  the  crouching 
position  upon  a  table.  If  the  animal  does  not  remain 
quiet  it  is  best  to  wrap  it  in  a  towel  so  that  nothing  but 
its  head  protrudes,  though  in  the  most  cases  we  have 
not  found  this  necessary,  and  particularly  if  the  animal 
has  not  been  excited  prior  to  the  beginning  of  the 
operation. 

The  animal  should  be  placed  so  that  the  ear  upon 


INJECTION    INTO    THE    CIRCULATION.       157 

which  the  operation  is  to  be  performed  comes  between 
the  operator  and  the  source  of  light.  This  renders 
visible  by  transmitted  light  not  only  the  coarser  vessels 
of  the  ear,  but  also  their  finer  branches.  The  point  at 
which  the  injection  is  to  be  made  is  to  be  shaved  clean 
of  hair,  by  means  of  a  razor  and  soap. 

The  filled  hypodermatic  syringe  is  taken  in  one  hand 
and  with  the  other  hand  the  ear  is  held  firmly.  The 
point  of  the  needle  is  then  inserted  through  the  skin 
and  into  the  finest  part  of  the  ramus  posterior,  the  part 
nearest  the  apex  of  the  ear.  When  the  point  of  the 
needle  is  in  this  vessel  it  gives  to  the  hand  a  sensation 
quite  different  from  that  felt  when  it  is  in  the  midst  of 
connective  tissue.  As  soon  as  one  thinks  the  point  of 
the  needle  is  in  the  vessel,  a  drop  or  two  of  the  fluid 
may  be  injected  from  the  syringe,  and  if  his  suspicions 
are  correct  the  circulation  in  the  small  ramifications  and 
their  anastomoses  will  quickly  alter  in  appearance. 
Instead  of  their  containing  blood,  the  colorless  fluid 
which  is  being  injected  will  now  be  seen  to  circulate. 
This  must  be  carefully  observed,  for  sometimes  when  the 
needle-point  is  not  actually  in  the  vessel,  but  is  in  the 
lymph-spaces  surrounding  it,  an  appearance  somewhat 
similar  is  to  be  seen.  It  may  always  be  differentiated, 
however,  by  continuing  the  injection,  when  the  circu- 
lation of  clear  fluid  through  the  vessels  will  not  only 
fail  to  take  the  place  of  the  circulating  blood  but 
there  will  at  the  same  time  appear  a  localized  swelling 
under  the  skin  about  the  point  of  the  needle.  The 
needle  must  then  be  withdrawn  and  inserted  into  the 
vessel  at  a  point  a  little  nearer  to  its  proximal  end. 

Care  must  be  given  that  no  air  is  injected. 

The  hypodermatic  syringe  and  needle  must,  previous 


158 


BACTERIOLOGY. 


to  operation,  have  been  carefully  sterilized  in  the  steam 
sterilizer.  The  animal  must  be  kept  under  close  observa- 
tion for  about  an  hour  after  injection. 

The  form  of  syringe  best  suited  for  this  operation  is 
of  the  ordinary  design,  but  one  that  permits  of  thorough 
sterilization  by  steam.  It  should  be  made  of  glass  and 
metal,  with  asbestos  packings.  The  syringes  commonly 
employed  are  those  shown  in  Fig.  25 — A,  Koch's ;  J5, 
Strohschein's ;  (7,  Overlack's. 

FIG.  25. 


The  operation  is  one  that  cannot  be  learned  from 
verbal  description.  It  can  only  be  successfully  per- 
formed after  actual  practice. 

If  the  precautions  which  have  been  mentioned  are 
observed,  but  little  difficulty  in  performing  the  opera- 
tion will  be  experienced. 

Its  convenience  and  simplicity  over  other  methods  for 
the  introduction  of  substances  into  the  circulation  com- 
mend it  as  an  operation  with  which  to  make  oneself 
familiar.  The  animals  sustain  practically  no  wound, 
they  experience  no  pain — at  least  they  give  no  evidence 
of  pain — and  no  anaesthesia  is  required. 


CHAPTER    XIV. 

Post-mortem  examination  of  animals — Bacteriological  examina- 
tion of  the  tissues — Disposal  of  tissues  and  disinfection  of  instru- 
ments after  the  examination. 

FOR  the  purpose  of  examining  bacteriological  ly  the 
tissues  of  dead  animals,  certain  rigid  precautions  must 
be  observed  in  order  to  avoid  error. 

The  autopsy  should  be  made  as  soon  as  possible  after 
death.  If  delay  cannot  be  avoided,  the  animal  should 
be  kept  on  ice  until  the  examination  can  be  made,  other- 
wise decomposition  sets  in,  and  the  saprophytic  bacteria 
which  will  now  be  present  may  interfere  with  the  accu- 
racy of  the  results. 

When  the  autopsy  is  to  be  made,  the  animal  is 
first  inspected  externally,  and  all  visible  lesions  noted. 
It  is  then  to  be  fixed  upon  its  back  upon  a  board  with 
nails  or  tacks.  The  four  legs  and  the  end  of  the  nose, 
through  which  the  tacks  are  driven,  are  to  be  moderately 
extended.  The  surfaces  of  the  thorax  and  abdomen  are 
then  to  be  moistened  to  prevent  the  fine  hairs,  dust,  etc., 
from  floating  about  in  the  air  and  interfering  with  the 
work.  An  incision  is  then  made  through  the  skin  from 
the  chin  to  the  genitalia.  This  is  only  a  skin  incision,  and 
does  not  reach  deeper  than  the  muscles.  It  is  best  clone 
by  first  making  a  small  incision  with  a  scalpel,  just  large 
enough  to  permit  of  the  introduction  of  one  blade  of  a 
blunt-pointed  scissors.  It  is  then  completed  with  the 
scissors.  The  whole  of  the  skin  is  then  carefully  dissected 


160  BACTERIOLOGY. 

away,  not  only  from  the  abdomen  and  thorax,  but  from 
the  axillary,  inguinal,  and  cervical  regions,  and  the  fore 
and  hind  legs  as  well.  The  skin  is  then  pinned  back  to 
the  board  so  as  to  keep  it  as  far  from  the  abdomen  and 
thorax  as  possible,  for  it  is  from  the  skin  that  the 
chances  of  contamination  are  greatest. 

It  now  becomes  necessary  to  proceed  very  carefully. 
All  incisions  from  this  time  on  are  to  be  made  only 
through  surfaces  that  have  been  sterilized.  This  is  best 
accomplished  by  the  use  of  a  broad-bladed  common  knife 
which  can  be  heated  in  the  gas-flame.  The  blade  is  to  be 
heated  quite  hot,  and  is  to  be  held  upon  the  region  of 
the  linea  alba  until  the  skin  at  that  region  begins  to 
burn  ;  it  is  then  held  transverse  to  this  line  over  about 
the  centre  of  the  abdomen,  thus  making  two  sterilized 
tracks  through  which  the  abdomen  may  be  opened  by  a 
crucial  incision.  The  sterilization  thus  accomplished  is, 
of  course,  directed  only  against  organisms  that  may  have 
fallen  upon  the  surface  from  without,  and  it  therefore 
need  not  extend  deep  down  through  the  tissues. 

In  the  same  way  two  burned  lines  may  be  made  from 
either  extremity  of  the  transverse  line  up  to  the  top  of 
the  thorax. 

With  a  hot  scissors  the  central  longitudinal  incision, 
extending  from  the  point  of  the  sternum  to  the  geuitalia, 
is  to  be  made  without  touching  the  internal  viscera.  The 
abdominal  wall  must  therefore  be  held  up  during  the 
operation  with  sterilized  forceps  or  hook. 

The  cross  incision  is  made  in  the  same  way.  When 
this  is  completed,  an  incision  through  the  ribs  with  a  pair 
of  heavy  scissors,  which  have  been  sterilized,  is  made 
along  the  scorched  tracks  on  either  side  of  the  thorax. 

After  this  the  whole  anterior  wall  of  the  thorax  may 


EXAMINATION    OF    ANIMALS.  161 

easily  be  lifted  up,  and  by  severing  the  connections  with 
the  diaphragm  it  may  be  completely  removed. 

When  this  is  done  and  the  abdominal  flaps  laid  back, 
the  contents  of  both  cavities  are  to  be  inspected  and  their 
condition  noted  without  disturbing  them. 

After  this,  the  first  steps  to  be  taken  are  to  prepare 
plates  or  Esrnarch  tubes  from  the  point  of  inoculation, 
the  blood,  liver,  spleen,  kidneys,  and  any  exudates  that 
may  exist. 

This  is  best  done  as  follows : 

Heat  a  scalpel  quite  hot  and  apply  it  to  a  small  sur- 
face of  the  organ  from  which  the  cultures  are  to  be 
made.  Hold  it  upon  the  organ  until  the  surface  directly 
beneath  it  is  visibly  scorched.  Then  remove  it,  heat  it 
again  and  while  quite  hot  insert  its  point  through  the 
capsule  of  the  organ.  Into  the  opening  thus  made  insert 
a  sterilized  platinum-wire  loop,  made  of  wire  a  little 
heavier  than  that  commonly  employed.  Project  this 
deeply  into  the  tissues  of  the  organ ;  by  twisting 
it  about,  enough  material  from  the  centre  of  the  organ 
can  be  obtained  for  making  plates  or  Esmarch  tubes. 

The  cultures  from  the  blood  are  usually  made  from 
one  of  the  cavities  of  the  heart,  which  is  always  entered 
through  a  surface  which  has  been  burned  in  the  way 
given. 

In  addition  to  cultures,  cover-slips  from  each  organ 
and  from  any  exudates  that  may  exist,  must  be  made. 
These,  however,  are  prepared  after  the  materials  for  the 
cultures  have  been  obtained. 

They  need  not  be  examined  immediately,  but  may  be 
placed  aside,  under  cover,  on  bits  of  paper  upon  which 
the  name  of  the  organ  from  which  they  were  prepared 
is  written. 


162  BACTERIOLOGY. 

When  the  autopsy  is  complete  and  the  gross  appear- 
ances have  been  carefully  noted,  small  portions  of  each 
organ  are  to  be  preserved  in  95  per  cent,  alcohol  for  sub- 
sequent examination.  Throughout  the  entire  autopsy  it 
must  be  borne  in  mind  that  all  cultures,  cover-slips,  and 
tissues  must  be  carefully  labelled,  not  only  with  the 
name  of  the  organ  from  which  they  originate,  but  with 
the  date,  name  of  the  animal,  etc.,  so  that  an  account  of 
their  condition  after  closer  study  may  be  subsequently 
inserted  in  the  protocol. 

The  cover-slips  are  now  to  be  stained,  mounted,  and 
examined  microscopically,  and  the  results  carefully  noted 
in  the  protocol. 

The  same  maybe  said  for  the  subsequent  study  of  the 
cultures  and  the  hardened  tissues  which  are  to  be  stained 
and  subjected  to  microscopic  examination.  The  results 
of  the  microscopic  study  of  the  cover-slip  preparations 
and  those  obtained  by  cultures  should  in  most  cases  corre- 
spond, though  it  not  rarely  occurs  that  bacteria  are  present 
in  such  small  numbers  in  the  tissues  that  their  presence 
may  be  overlooked  microscopically,  and  still  they  may 
appear  in  the  cultures. 

If  the  autopsy  has  been  performed  in  the  proper  way, 
under  the  precautions  given,  and  sufficiently  soon  after 
death,  the  results  of  the  bacteriological  examinatiou 
should  be  either  negative  or  the  organisms  which 
appear  should  be  in  pure  cultures. 

This  is  particularly  the  case  with  the  cultures  made 
from  the  internal  viscera. 

Both  the  cover-slips  and  cultures  made  from  the  point 
of  inoculation  are  apt  to  contain  a  variety  of  organisms. 

If  the  organism  obtained  in  pure  culture  from  the  in- 
ternal viscera,  or  those  predominating  at  the  point  of 


EXAMINATION    OF    ANIMALS.  163 

iooculation  of  the  animal,  have  caused  its  death,  then 
subsequent  inoculation  of  pure  cultures  of  this  organ- 
ism into  the  tissues  of  a  second  animal  should  produce 
similar  results. 

When  the  autopsy  is  quite  finished,  the  remainder  of 
the  animal  should  be  burned,  all  instruments  subjected 
to  either  sterilization  by  steam  or  boiling  for  fifteen 
minutes  in  1  to  2  per  cent,  soda  solution,  and  the  board 
upon  which  the  animal  was  tacked,  as  well  as  the  tacks, 
towels,  dishes,  and  all  other  implements  used  at  the 
autopsy,  are  to  be  sterilized  by  steam.  All  cultures, 
cover-slips,  and  indeed,  all  articles  likely  to  have  in- 
fectious material  upon  them,  must  be  thoroughly  steril- 
ized as  soon  as  they  are  of  no  further  service. 


CHAPTER   XV. 

Scheme  for  the  complete  study  of  an  organism. 

THE  following  scheme  will  serve  as  a  guide  for  the 
systematic  study  of  au  organism  : 

1.  Its  form  and  grouping  as  seen  when  discovered. 

2.  Where  discovered. 

3.  The  appearance  of  its  colonies  on  gelatin  plates  or 
Esmarch  tubes. 

4.  The  appearance  of  its  colonies  on  agar-agar  plates 
or  Esmarch  tubes. 

5.  The  appearance  of  its  growth  in  stab  and  slant 
cultures  on  gelatin. 

6.  The  appearance  of  its  growth  in  stab  and  slant 
cultures  on  agar-agar. 

7.  Its  growth  on  potato. 

8.  Its  growth  on  blood-serum. 

9.  Its  behavior  in  bouillon. 

10.  Its  behavior  in  milk,  plain. 

11.  Its  behavior  in  peptone- rosolic-acid  solution. 

12.  Its  behavior  in  milk  containing  litmus  solution. 

13.  What  is   its  normal   morphology?     What  mor- 
phological changes  does  it  pass  through  under  varying 
conditions  of  life? 

14.  Does  it  form  spores  ? 

15.  Is  it  motile?     Are  the  flagelhe  demonstrable  by 
Loffler's  method  of  staining?     Is  an  acid  or  an  alkali 
to  be  added  to  the  mordant ;  if  so,  the  quantity  ?     In 


SCHEME    FOR    STUDY    OF    AN    ORGANISM.       165 

what  way  are  the  flagellse  given  off  from  the  body  of  the 
organism  ? 

16.  Does  it  produce  gas-bubbles  in  ordinary  agar- 
agar  or  gelatin  ? 

17.  Does  it  produce  gas-bubbles  in  agar-agar  or  gel- 
atin to  which  1  to  2  per  cent,  grape  sugar  has  been 
added  ? 

18.  Does   it   produce   indol?     Is   the   iudol   accom- 
panied by  a  coincident  production  of  nitrites  ? 

19.  At  what  temperature  does  it  grow  most  luxu- 
riantly ? 

20.  What  is    the    lowest   temperature   at    which    it 
grows  ? 

21.  Is  it  aerobic,  anaerobic,  or  facultative  in  its  rela- 
tions to  oxygen? 

22.  What  are  its  staining  peculiarities  ? 

23.  Will  it  withstand  drying? 

24.  At  what  temperature  is  it  killed  by  heat — both 
by  steam  and  the  hot-air  method  ? 

25.  Is  it  pathogenic  when  introduced  either  subcu- 
taneously  or  directly  into  the  circulation  of  animals? 
If  so,  for  which  animals?     Do  any  of  the  animals  used 
for  this  work  possess  natural  immunity  against  infection 
by  this  organism  ? 

26.  What  are  the  histological  appearances  seen  in  the 
tissues  of  animals  for  which  this  organism  is  pathogenic  ? 


PRACTICAL  APPLICATION  OF  THE  METHODS 
OF  BACTERIOLOGY. 


CHAPTER  XVI. 

To  obtain  material  upon  which  to  begin  work. 

EXPOSE  to  the  air  of  an  inhabited  room  a  slice  of 
freshly  steamed  potato  or  a  bit  of  slightly  moistened 
bread  upon  a  plate  for  about  one  hour.  Then  cover  it 
with  an  ordinary  water-glass  and  place  it  in  a  warm  spot 
(temperature  not  to  exceed  that  of  the  human  body — 
37.5°  C.),  and  allow  it  to  remain  unmolested.  At  the 
end  of  twenty-four  to  thirty-six  hours  there  can  be 
seen  upon  the  cut  surface  of  the  bread  or  potato  small, 
round,  oval,  or  irregularly  round  patches  which  present 
various  appearances. 

These  differences  in  macroscopic  appearance  con- 
sist in  some  cases  in  the  presence  or  absence  of  color ; 
again  in  a  higher  or  lower  degree  of  moisture;  in  some 
instances  a  patch  will  be  glistening  and  smooth  while  its 
neighbor  may  be  dull  and  rough  or  wrinkled.  Here 
will  appear  an  island  regularly  round  in  outline  and 
there  an  area  covered  by  an  irregular  ragged  deposit. 
All  of  these  gross  appearances  are  of  value  in  aiding  us 
to  distinguish  between  these  colonies — for  colonies  they 
are — and  under  the  same  conditions  the  organisms  com- 


168  BACTERIOLOGY. 

posing  each  of  them  will  always  produce  growths  of  ex- 
actly the  same  appearance.  It  was  just  such  an  experi- 
ment as  this,  accidentally  performed,  that  suggested  to 
Koch  a  means  of  separating  and  isolating  from  mix- 
tures of  bacteria  the  component  individuals  in  pure 
cultures,  and  it  is  upon  this  observation  that  the 
methods  of  cultivation  on  solid  media  are  based. 

If,  without  molesting  our  experiment,  we  continue  the 
observation  from  day  to  day,  we  may  notice  changes  in 
the  colonies  due  to  the  growth  and  multiplication  of  the 
individuals  composing  them.  In  some  cases  the  colo- 
nies will  always  retain  their  sharply  cut,  round,  or  oval 
outline,  and  will  increase  but  little  in  size  beyond  that 
reached  after  forty-eight  to  seventy-two  hours,  whereas 
others  will  spread  rapidly,  and  will  very  quickly  over- 
run the  surface  upon  which  they  are  growing,  and  in- 
deed, grow  over  the  smaller,  less  rapidly  developing 
colonies.  In  a  number  of  instances,  if  the  observation 
be  continued  long  enough,  many  of  these  rapidly  grow- 
ing colonies  will,  after  a  time,  lose  their  lustrous  and 
smooth  or  regular  surface  and  will  show,  at  first  here  and 
there,  elevations  which  will  continue  to  appear  until  the 
whole  surface  takes  on  a  wrinkled  appearance.  Again 
bubbles  may  be  seen  here  and  there  through  the  colo- 
nies. These  are  due  to  the  escape  of  gas  resulting  from 
fermentation  which  the  organisms  bring  about  in  the 
medium  upon  which  they  are  growing.  Sometimes 
peculiar  odors  resulting  from  the  same  cause  will  be 
noticed. 

Note  carefully  all  these  changes  and  appearances,  as 
they  must  be  employed  subsequently  in  identifying  the 
individual  organisms  from  which  each  colony  on  the 
medium  is  growing. 


EXPOSUEE    AND    CONTACT.  169 

If  now  we  examine  these  points  upon  our  bread  or 
potato  with  a  hand-lens  of  low  magnifying  power  we 
will  be  enabled  to  detect  differences  not  noticeable  to  the 
naked  eye.  In  some  cases  we  shall  still  see  nothing  more 
than  a  smooth  non-characteristic  surface;  while  in  others, 
minute,  sometimes  regularly  arranged  corrugations  may 
be  observed.  In  one  colony  they  may  appear  as  toler- 
ably regular  radii,  radiating  from  a  central  spot ;  and 
again  they  may  appear  as  concentric  rings ;  and  if  by 
the  methods  which  have  been  described  we  obtain  from 
these  colonies  their  individual  components  in  pure  cul- 
ture, we  shall  see  that  this  characteristic  arrangement 
in  folds,  radii,  or  concentric  rings,  or  the  production  of 
color,  is  under  normal  conditions  constant. 

So  much  for  the  simplest  naked-eye  experiment  that 
can  be  made  in  bacteriology  and  which  serves  to  furnish 
the  beginner  with  material  upon  which  to  begin  his 
studies.  It  is  not  necessary  at  this  time  for  him  to 
burden  his  mind  with  names  for  these  organisms;  it  is 
sufficient  for  him  to  recognize  that  they  are  mostly  of 
different  species  and  that  they  possess  characteristics 
which  will  enable  him  to  differentiate  the  one  from  the 
other. 

In  order  now  for  him  to  proceed  it  is  necessary  that 
he  should  have  familiarized  himself  with  the  methods 
by  which  his  media  are  prepared  and  the  means  em- 
ployed in  sterilizing  them  and  retaining  them  sterile — 
i.  e.,  of  preventing  the  access  of  foreign  germs  from 
without — otherwise  his  efforts  to  obtain  and  retain  his 
organisms  as  pure  cultures  will  be  in  vain. 

EXPOSURE  AND  CONTACT. — Make  a  number  of  plates 
from  bits  of  silk  used  for  sutures,  after  treating  them 
as  follows  : 


170  BACTERIOLOGY. 

Place  some  of  these  pieces  (about  5  centimetres  long) 
into  a  sterilized  test-tube,  and  sterilize  them  by  steam 
for  one  hour.  At  the  end  of  the  sterilization  remove 
one  piece  with  sterilized  forceps  and  allow  it  to  brush 
against  your  clothing,  then  make  a  plate  from  it ;  an- 
other piece  draw  across  the  table  and  then  plate  it.  Sus- 
pend upon  a  sterilized  wire  hook  three  or  four  pieces 
and  let  them  hang  for  thirty  mintucs  free  in  the  air, 
being  sure  that  they  touch  nothing  but  the  hook  ;  then 
plate  them  separately. 

Note  the  results. 

In  what  way  do  these  experiments  differ  and  how  can 
the  differences  be  explained  ? 

Expose  to  the  air  six  Petri  dishes  into  which  either 
sterilized  gelatin  or  agar-agar  has  been  poured  and  allowed 
to  solidify  ;  allow  them  to  remain  exposed  for  five,  ten, 
fifteen,  twenty,  twenty-five,  and  thirty  minutes,  in  a 
room  where  no  one  is  at  work.  Treat  a  second  set  in 
the  same  way  in  a  room  where  several  persons  are 
moving  about.  Be  careful  that  nothing  touches  them, 
and  that  they  are  exposed  only  to  the  air.  Each  dish 
must  be  carefully  labled  with  the  time  of  its  exposure. 

Do  they  present  different  results?  What  is  the  rea- 
son for  this  difference  ? 

Which  predominate,  colonies  resulting  from  the 
growth  of  bacteria,  or  those  from  common  moulds? 

How  do  you  account  for  this  condition  ? 


CHAPTER   XVII. 

Various  experiments  in  sterilization — Steam  and  hot-air  methods 
of  sterilizing. 

PLACE  in  one  of  the.  openings  in  the  cover  of  the  steam 
sterilizer  an  accurate  thermometer ;  when  the  steam  has 
been  streaming  for  a  minute  or  two  the  thermometer  will 
register  100°  C. ;  wrap  in  a  bundle  of  towels  or  rags  or 
pack  tightly  in  cotton  a  maximum  thermometer ;  let  this 
thermometer  be  in  the  centre  of  a  bundle  large  enough 
to  quite  fill  the  chamber  of  the  sterilizer.  At  the  end 
of  a  few  minutes  exposure  to  the  streaming  steam 
remove  it;  it  will  be  found  to  indicate  a  tempera- 
ture of  100°  C. 

Closer  study  of  the  penetration  of  steam  has  taught 
us,  however,  that  the  temperature  which  is  found  at  the 
centre  of  such  a  mass  may  sometimes  be  that  of  the 
air  in  the  meshes  of  the  material,  and  not  that  of 
steam,  and  for  this  reason  the  sterilization  at  that 
point  may  not  be  complete,  because  hot  air  at  100°  C. 
has  not  the  destructive  properties  that  steam  at  the 
same  temperature  possesses.  It  is  necessary,  there- 
fore, that  this  air  should  be  expelled  from  the  meshes 
of  the  material  and  its  place  taken  by  the  steam  be- 
fore sterilization  is  complete.  This  is  insured  by  allow- 
ing the  steam  to  stream  through  the  substances  a  few 
minutes  before  beginning  to  calculate  the  time  of  ex- 
posure. There  is  as  yet  no  absolutely  sure  means  of 
saying  that  the  temperature  at  the  centre  of  the  mass  is 
that  of  hot  air  or  of  steam,  so  that  the  exact  length  of 


172  BACTERIOLOGY. 

time  that  is  required  for  the  expulsion  of  the  air  from 
the  meshes  of  the  material  cannot  be  given. 

Determine  if  the  maximum  thermometer  indicates  a 
temperature  of  100°  C.  at  the  centre  of  a  moist  bundle 
in  the  same  way  as  when  a  dry  bundle  was  employed. 

To  about  50  c.c.  of  bouillon  add  about  one  gramme  of 
chopped  hay,  and  allow  it  to  stand  in  a  warm  place  for 
twenty-four  hours.  At  the  end  of  this  time  it  will  be 
found  to  contain  a  great  variety  of  organisms.  Continue 
the  observation,  and  a  pellicle  will  be  seen  to  form  on 
the  surface  of  the  fluid.  This  pellicle  will  be  made  up 
of  rods  which  grow  as  long  threads  in  parallel  strands. 
In  many  of  these  rods  glistening  spores  will  be  seen. 
After  thoroughly  shaking,  filter  the  mass  through  a  fine 
cloth  to  remove  coarser  particles. 

Pour  into  each  of  several  test-tubes  about  10  c.c.  of 
the  filtrate.  Allow  one  tube  to  remain  unmolested  in 
a  warm  place.  Place  another  in  the  steam  sterilizer  for 
five  minutes.  A  third  should  remain  for  ten  minutes. 
A  fourth  for  one-half  hour.  A  fifth  for  one  hour. 

At  the  end  of  each  of  these  exposures  inoculate  a  tube 
of  sterilized  bouillon  from  each  tube.  Likewise  make 
a  set  of  plates  or  Esmarch  tubes  upon  both  gelatin  and 
agar-agar  from  each  tube,  and  note  the  results.  At  the 
same  time  prepare  a  set  of  plates  or  Esmarch  tubes  on 
agar-agar  and  on  gelatin  from  the  tube  which  has  not 
been  exposed  to  the  action  of  the  steam. 

The  plates  or  tubes  from  the  unmolested  tube  will 
present  colonies  of  a  variety  of  organisms ;  separate  and 
study  these. 

Those  from  the  tube  which  has  been  sterilized  for  five 
minutes  will  present  colonies  in  moderate  numbers,  but, 


STEAM    AND    HOT-AIR    STERILIZING.      173 

as  a  rule,  they  will  represent  but  a  single  organism. 
Study  this  organism  in  pure  cultures. 

The  same  may  be  predicted  for  the  tube  which  has 
been  heated  for  ten  minutes,  though  the  colonies  will  be 
fewer  in  number. 

The  thirty-minute  tube  may  or  may  not  give  one  or 
two  colonies  of  the  same  organism. 

The  tube  which  has  been  heated  for  one  hour  is 
usually  sterile. 

The  bouillon  tubes  from  the  first  and  second  tubes 
which  were  heated  will  usually  show  the  presence  of 
only  one  organism — the  bacillus  which  gave  rise  to  the 
pellicle- formation  in  our  original  mixture.  This  organ- 
ism is  the  bacillus  subtilis,  and  will  serve  to  illustrate  the 
difference  in  resistance  toward  steam  between  the  vege- 
tative and  spore  stages  of  the  same  organism. 

Inoculate  about  100  c.c.  of  sterilized  bouillon  with  a 
very  small  quantity  of  a  pure  culture  of  this  organism, 
and  allow  it  to  stand  in  a  warm  place  for  about  six 
hours.  Now  subject  this  culture  to  the  action  of  steam 
for  five  minutes ;  it  will  be  seen  that  sterilization,  as  a 
rule,  is  complete. 

Treat  in  the  same  way  a  second  flask  of  bouillon,  in- 
oculated in  the  same  way  with  the  same  organism,  but 
after  having  stood  in  a  warm  place  for  from  forty-eight 
to  seventy -two  hours,  that  is,  until  the  spores  have 
formed,  and  it  will  be  found  that  sterilization  is  not 
complete — the  spores  of  this  organism  have  resisted 
the  action  of  steam  for  five  minutes. 

To  determine  if  sterilization  is  complete  always  resort 
to  the  culture  methods,  as  the  macroscopic  and  micro- 
scopic methods  are  deceptive.  Cloudiness  of  the  media 
or  the  presence  of  organisms  microscopically  does  not 


174  BACTERIOLOGY. 

always  signify  that  the  organisms  possess  the  property 
of  life. 

Inoculate  in  the  same  way  a  third  flask  of  bouillon 
with  a  very  small  drop  from  one  of  the  old  cultures 
upon  which  the  pellicle  has  formed  ;  mix  it  well  and 
subject  it  to  the  action  of  steam  for  two  minutes ;  then 
place  it  to  one  side  for  from  twenty  to  twenty-four 
hours,  and  again  heat  for  two  minutes;  allow  it  to  stand 
for  another  twenty-four  hours,  and  repeat  the  process  on 
the  third  day.  No  pellicle  will  be  formed,  and  yet  spores 
were  present  in  the  original  mixture,  and,  as  we  have 
seen,  the  spores  of  this  organism  are  not  killed  by  an 
exposure  of  five  minutes  to  the  steam.  How  can  this 
result  be  accounted  for  ? 

Saturate  several  pieces  of  cotton  thread,  each  about 
2  cm.  long,  in  the  original  decomposed  bouillon,  and  dry 
them  carefully  at  the  ordinary  temperature  of  the  room, 
then  at  a  little  higher  temperature — about  40°  C. — to 
complete  the  process.  Regulate  the  temperature  of  the 
hot-air  sterilizer  for  about  100°  C.,  and  subject  several 
pieces  of  this  infected  and  dried  thread  to  this  tempera- 
ture for  the  same  lengths  of  time  that  we  exposed  the 
same  organisms  in  bouillon  to  the  steam,  viz.  :  five,  ten, 
thirty,  and  sixty  minutes.  At  the  end  of  each  of  these 
periods  remove  a  bit  of  thread,  and  prepare  a  set  of  plates 
or  Esmarch  tubes  from  it.  Are  the  results  analogous  to 
those  obtained  when  steam  was  employed  ? 

Increase  the  temperature  of  the  dry  sterilizer  and 
repeat  the  process.  Determine  the  temperature  and 
time  necessary  for  the  destruction  of  these  organisms 
by  the  dry  heat.  These  threads  should  not  be  simply 
laid  upon  the  bottom  of  the  sterilizer,  but  should  be  sus- 


STEAM    AND    HOT-AIR     STERILIZING.      175 

pended  from  a  glass  rod,  which  may  be  placed  inside 
the  oven,  extending  across  its  top  from  one  side  to  the 
other. 

Place  several  of  the  infected  threads  in  the  centre  of 
a  bundle  of  rags.  Subject  this  to  a  temperature  necessary 
to  sterilize  the  threads  by  the  dry  method.  Treat 
another  similar  bundle  to  sterilization  by  steam.  In 
what  way  do  the  two  processes  differ? 


CHAPTER    XVIII. 

Bacteriological  study  of  water,  air,  and  soil — Methods  of  counting 
the  colonies  on  the  plates — Wolff  hiigel's  counting  apparatus — Sedg- 
wick's  method. 

THE  possible  spread  of  infectious  diseases  by  means 
of  water-supplies  has  formed  a  topic  of  discussion  by 
sanitarians  for  a  long  time. 

The  school  of  Von  Pettenkofer  has  always  taken  the 
ground  that  the  appearance  and  spread  of  epidemic 
diseases,  of  which  typhoid  fever  and  Asiatic  cholera  are 
types,  is  due  more  to  alterations  in  the  soil,  resulting 
from  fluctuations  in  the  level  of  the  soil-water,  than 
to  any  part  that  the  drinking-water  may  play ;  in  op- 
position to  this  Koch  and  his  pupils  hold  that  these 
epidemics  can,  in  most  instances,  be  traced  directly  to  the 
influence  of  the  water-supply. 

The  weight  of  evidence  as  it  now  stands  favors  the 
opinion  that  these  diseases  are  frequently  the  result  of 
imperfections  in  the  supply  of  water  intended  for  do- 
mestic purposes,  and  there  exists  sufficient  proof  of  this 
to  necessitate  our  controlling  all  such  supplies  by  careful 
quantitative  and  qualitative  bacteriological  analyses. 

THE  QUALITATIVE  BACTERIOLOGICAL  ANALYSIS  OF 
WATER.  —  The  qualitative  bacteriological  analysis  of 
water  entails  much  labor,  as  it  requires  that  not  only 
all  the  different  species  of  organism  found  in  the  water 
should  be  isolated,  but  that  each  representative  should 
be  subjected  to  systematic  study,  and  its  pathogenic  or 
non-pathogenic  properties  determined. 


ANALYSIS    OF    WATER.  177 

For  this  purpose  the  methods  for  the  isolation  of 
individual  species,  which  have  already  been  described, 
and  the  means  of  studying  these  species  when  isolated, 
are  indispensable. 

For  this  analysis  certain  precautions  essential  to 
accuracy  are  always  to  be  observed. 

The  sample  is  to  be  collected  under  the  most  rigid 
precautions  that  will  exclude  organisms  from  sources 
other  than  that  under  consideration.  If  drawn  from  a 
spigot,  it  should  never  be  collected  until  the  water  has 
been  flowing  for  15  to  20  minutes  in  a  full  stream.  If 
obtained  from  a  stream  or  a  spring,  it  should  be  collected, 
not  from  the  surface,  but  rather  from  about  one  foot 
beneath  the  surface. 

It  should  always  be  collected  in  vessels  which  have 
previously  been  thoroughly  freed  from  all  dirt  and 
organic  particles,  and  then  sterilized.  And  the  plates 
should  be  made  as  quickly  after  collecting  the  sample  as 
is  possible. 

Where  circumstances  permit,  all  water  analyses  should 
be  made  on  the  spot  at  which  the  sample  is  taken,  as  it 
is  known  that  during  transportation,  unless  the  samples 
are  kept  packed  in  ice,  a  multiplication  of  the  organisms 
contained  in  it  always  occurs. 

It  is  therefore  advisable  that  where  this  work  is  to  be 
done,  the  Esmarch  tubes  or  Petri  plates  should  be  pre- 
pared on  the  spot. 

For  the  purpose  of  qualitative  analysis  it  is  necessary 
that  a  small  portion  of  the  water — one,  two,  three,  five 
drops — should  first  be  employed  as  the  amounts  from 
which  plates  are  to  be  made.  In  this  way  one  forms 
some  idea  as  to  the  approximate  number  of  organisms 


178  BACTERIOLOGY. 

in  the  water,  and   can    in  consequence  determine  the 
amount  of  water  necessary  to  use  for  each  set  of  plates. 

Duplicate  plates  are  always  to  be  made — one  set  upon 
agar-agar,  which  are  to  be  kept  at  the  body -temperature, 
and  one  set  upon  gelatin,  which  are  to  be  kept  at  a  tem- 
perature of  18°  to  20°  C. 

As  soon  as  the  colonies  have  developed,  the  plates  are 
to  be  carefully  compared  and  studied.  It  is  to  be  noted 
if  any  difference  in  the  appearance  and  number  of 
organisms  on  corresponding  plates  exist,  and  if  so  on 
which  plates  the  larger  number  of  colonies  have  de- 
veloped. In  this  way  the  temperature  most  favorable 
for  the  growth  of  most  of  these  organisms  may  be 
determined.  The  opinion  has  been  advanced  that  many 
of  the  organisms  constantly  present  in  water,  which 
make  up  its  normal  flora,  develop  better  at  a  lower  than 
at  a  higher  temperature.  This  will  not  be  the  case, 
however,  if  pathogenic  forms  are  present,  because  they, 
as  a  rule,  require  the  body-temperature  for  their  most 
favorable  development,  though  some  of  them  do  grow 
very  well  at  a  lower  temperature. 

The  isolation  of  the  different  species  and  their  sys- 
tematic study  is  to  be  conducted  in  the  way  given  for  all 
bacteria. 

THE  QUANTITATIVE  ESTIMATION  OF  BACTERIA  IN 
WATER. — The  quantitative  analysis  requires  more  care  in 
the  measurement  of  the  exact  volume  of  water  employed, 
for  the  results  are  to  be  expressed  in  terms  of  the  number 
of  individual  organisms  to  a  definite  volume.  The 
necessity  for  making  the  plates  at  the  place  at  which  the 
sample  is  collected  is  to  be  particularly  accentuated  in 
this  analysis,  for  the  multiplication  of  the  organisms 
during  transit  is  so  great  that  the  results  of  analyses 


ANALYSIS    OF    WATER.  179 

made  after  the  water  has  been  in  a  vessel  for  a  day  or 
two  are  often  very  different  from  those  which  would 
have  been  obtained  on  the  spot. 

Where  it  is  not  possible,  however,  to  make  the  analysis 
on  the  spot,  the  sample  of  water  should  be  collected  and 
packed  in  ice  and  kept  on  ice  until  ready  for  use,  which 
should  in  all  cases  be  as  soon  after  its  collection  as 
possible. 

For  the  collection  of  water  for  this  purpose,  the  best 
vessel  to  be  employed  is  a  glass  bulb  (Fig.  26)  or 
balloon,  which  one  soon  learns  to  make  for  himself 
from  glass  tubing. 

It  consists  simply  of  a  round  glass  sphere  blown  on 
the  end  of  a  glass  tube,  which  latter  is  subsequently 

FIG.  26. 


drawn  out  into  a  fine  capillary  stem  and  sealed  while 
hot.  As  it  cools,  the  contraction  of  the  air  within  the 
bulb  results  in  the  production  of  a  negative  pressure.  If 
the  point  of  the  stem  be  broken  off  under  water,  the 
bulb  is  quickly  filled  because  of  the  existence  of  the 
negative  pressure  within  it. 

A  number  of  them  may  be  blown,  sealed,  and  kept 
on  hand.  They  are  sterile  so  long  as  they  are  sealed, 
because  of  the  heat  that  is  employed  in  their  manu- 
facture. 

When  a  sample  of  water  is  to  be  taken,  the  point  of 
a  bulb  is  simply  broken  off  with  sterilized  forceps  under 
water  at  the  place  from  which  the  sample  is  to  be  taken. 
It  rapidly  fills  with  water.  This  may  serve  as  a  sample. 


180  BACTERIOLOGY. 

from  which  to  prepare  plates  or  Esmarch  tubes  on  the 
spot,  or  the  tip  of  the  stem  may  be  re-sealed  in  the 
flame  of  an  alcohol  lamp,  the  bulb  packed  in  ice,  and 
transported  in  this  condition  to  the  laboratory. 

In  beginning  the  quantitative  analysis  of  water  with 
which  one  is  not  acquainted,  there  are  certain  preliminary 
steps  that  are  essential. 

It  is  necessary  to  know  approximately  the  number  of 
organisms  contained  in  any  fixed  volume,  so  as  to  de- 
termine the  quantity  of  water  to  be  employed  for  the 
plates  or  tubes.  This  is  done  usually  by  making  pre- 
liminary plates  from  one  drop,  two  drops,  0.25  c.c., 
0.5  c.c.,  and  1  c.c.  of  the  water.  After  each  plate  has 
been  labelled  with  the  amount  of  water  used  in  making 
it,  it  is  placed  aside  for  development.  When  this  has 
occurred,  one  selects  the  plate  upon  which  the  colonies 
are  only  moderate  in  number — about  200  to  300  colonies 
presenting — and  employs  in  the  subsequent  analysis  the 
same  amount  of  water  that  was  used  in  making  this 
plate. 

If  the  original  water  contained  so  many  organisms  that 
there  developed  on  a  plate  or  tube  made  with  one  drop  too 
many  colonies  to  be  easily  counted,  then  the  sample  must 
be  diluted  with  one,  two,  or  three  volumes,  as  the  case 
may  be,  of  sterilized  distilled  water.  This  dilution  must 
be  accurate,  and  its  exact  extent  noted,  so  that  subse- 
quently the  number  of  organisms  per  volume  in  the 
original  water  may  be  calculated. 

The  use  of  a  drop  is  not  sufficiently  accurate.  The 
dilution  should  therefore  always  be  to  a  degree  that  will 
admit  of  the  employment  of  a  volume  of  water  that  may 
be  exactly  measured,  0.25,  0.5  c.c.  being  the  amounts 
most  convenient  for  use. 


COUNTING    COLONIES    ON    PLATES.         181 

Duplicate  plates  should  always  be  made  and  the  mean 
of  the  number  of  colonies  that  develop  upon  them  taken 
as  the  basis  from  which  to  calculate  the  number  of 
organisms  per  volume  in  the  original  water. 

For  example :  From  a  sample  of  water,  0.25  c.c.  is 
added  to  a  tube  of  liquefied  gelatin,  carefully  mixed  and 
poured  out  as  a  plate.  When  development  occurs,  the 
number  of  colonies  are  too  numerous  to  be  accurately 
counted. 

One  cubic  centimetre  of  the  original  water  is  then  to 
have  added  to  it,  under  precautions  that  prevent  con- 
tamination from  without,  99  c.c.  of  sterilized  distilled 
water — that  is,  we  have  now  a  dilution  of  1  :  100. 
Again,  0.25  c.c.  of  this  dilution  is  plated  and  we  find 
180  colonies  on  the  plate.  Assuming  that  each  colony 
develops  from  an  individual  bacterium,  though  this  is  per- 
haps not  strictly  true,  we  had  180  organisms  in  0  25  c.  c. 
of  our  1  :  100  dilution,  therefore  in  0.25  c.c.  of  the 
original  water  we  had  180  X  100  =  18,000  bacteria, 
which  will  be  72,000  bacteria  per  cubic  centimetre  (0.25 
—  18,000,  1  c.c.  =  18,000  X  4  ==  72,000).  The  re- 
sults are  always  to  be  expressed  in  terms  of  the  number 
of  bacteria  per  cubic  centimetre  of  the  original  water. 

Throughout  this  part  of  the  work  it  is  to  be  borne  in 
mind  that  when  one  refers  to  plates  it  is  not  to  a  set,  as 
in  the  isolation  experiments,  but  to  a  single  plate. 

METHOD  OF  COUNTING  THE  COLONIES  ON  THE 
PLATES.  —  For  convenience  in  counting  colonies  on 
plates  or  in  tubes,  it  is  of  advantage  to  divide  the  whole 
area  of  the  gelatin  occupied  by  colonies  into  smaller  areas 
of  exact  size  For  this  purpose  several  very  convenient 
devices  exist. 


182  BACTERIOLOGY. 

WOLFFHUGEL'S  COUNTING  APPAEATUS. — This  ap- 
paratus (Fig.  27)  consists  of  a  flat  wooden  stand,  the 


FIG.  27. 


centre  of  which  is  cut  out  in  such  a  way  that  either  a 
black  or  white  glass  plate  may  be  placed  in  it.  These 
form  a  background  upon  which  the  colonies  may  more 
easily  be  seen  when  the  plate  to  be  counted  is  placed 
upon  it.  When  the  gelatin  plate  containing  the  colo- 
nies has  been  placed  upon  this  background  of  glass,  it  is 
then  covered  by  a  transparent  glass  plate  which  swings 
on  a  hinge.  When  this  plate  is  in  position,  it  is  just 
above  the  colonies  without  touching  them.  This  plate 
is  ruled  in  square  centimetres  and  subdivisions. 

The  gelatin  plate  is  moved  about  until  it  rests  under 
the  centre  of  the  area  occupied  by  the  ruled  lines. 

The  number  of  colonies  in  each  square  centimetre  is 
then  counted,  and  the  sum-total  of  the  colonies  in  all 
these  areas  gives  the  number  of  colonies  on  the  plate. 

Where  the  colonies  are  quite  small,  as  is  frequently 
the  case,  the  counting  may  be  facilitated  by  the  use  of  a 
small  hand-lens. 


ESMARCH'S  COUNTER.  183 

In  Fig.  28  is  seen  the  form  of  hand-lens  commonly 
employed. 

A  plan  that  is  frequently  given  for  the  counting  of 
colonies  by  the  use  of  these  devices  is  to  count  the  num- 
ber of  colonies  in  each  of  a  number  (eight  or  ten)  of  the 
squares,  and  take  the  average  of  these  counts  as  the 

FIG.  28. 


average  for  each  square  on  the  whole  surface  of  the 
gelatin.  The  result  is  then  obtained  by  multiplying 
this  average  by  the  number  of  squares  taken  up  by  the 
whole  surface  of  the  gelatin. 

The  results  vary  so  much  in  different  counts  of  the 
same  plate,  when  made  in  this  way,  that  they  can  hardly 
be  considered  approximate. 

Prepare  a  plate,  calculate  the  number  of  colonies  upon 
it  by  this  latter  method.  Now  repeat  the  calculation 
making  the  average  from  another  set  of  squares.  Now 
actually  count  the  entire  number  of  colonies  on  the  plate. 
Compare  the  results. 

ESMARCH'S  COUNTER. — Esmarch  has  devised  a  coun- 
ter (Fig.  29)  for  estimating  the  number  of  colonies  pres- 
ent when  they  are  upon  a  cylindrical  surface,  as  when  in 
rolled  tubes.  The  principles  and  methods  of  estimation 
are  practically  the  same  as*  those  given  for  Wolff  hiigePs 
apparatus.  If  the  number  of  colonies  in  an  Esmarch 
tube  is  to  be  determined,  a  simpler  method  to  the  use  of 


184  BACTERIOLOGY. 

his  apparatus  may  be  employed.  It  consists  in  dividing 
the  tube  by  lines  into  four  or  six  longitudinal  areas  which 
are  subdivided  by  transverse  lines  drawn  about  1  or 
2  cm.  apart  The  lines  may  be  drawn  with  pen  and 
ink.  They  need  not  be  exactly  the  same  distance  apart, 

FIG.  29. 


or  exactly  straight.  Beginning  at  one  of  these  squares 
at  one  end  of  the  tube,  which  may  be  marked  with  a 
cross,  the  tube  is  twisted  with  the  fingers,  always  in  one 
direction,  and  the  exact  number  of  colonies  in  each  square 
as  it  appears  in  rotation  is  counted,  care  being  taken  not 
to  count  a  square  more  than  once ;  they  are  then  added 
together,  and  the  result  gives  the  number  of  colonies  in 
the  tube.  This  method  may  be  facilitated  by  the  use  of 
a  hand-lens. 

In  all  these  methods  there  is  one  error  that  is  difficult 
to  eliminate ;  it  is  assumed  that  each  colony  represents 
the  outgrowth  from  a  single  organism.  This  is  prob- 


ESMARCH'S   COUNTER.  185 

ably  not  always  the  case,  as  there  may  exist  clumps  of 
bacteria  which  represent  hundreds  or  even  thousands  of 
individuals,  but  which  still  give  rise  to  but  a  single 
colony — this  is  usually  estimated  as  a  single  organism  in 
the  water  under  analysis. 

Where  grounds  exist  for  suspecting  the  presence  of 
these  clumps,  they  may  in  part  be  broken  up  by  shaking 
the  original  water  with  sterilized  sand. 

What  has  been  said  for  the  bacteriological  examina- 
tion of  water,  holds  good  for  all  fluids  which  are  to  be 
subjected  to  this  form  of  analysis. 

In  considering  water  from  a  bacteriological  standpoint, 
it  must  always  be  borne  in  mind  that  comparisons  of  the 
water  with  any  general  fixed  standard  are  not  of  much 
value,  for  just  as  normal  waters  from  different  sources  are 
seen  to  present  differences  in  their  chemical  composition 
without  being  unfit  for  use,  so  may  the  number  of 
bacteria  per  volume  in  water  from  one  source  always  be 
greater  o$  smaller  than  that  from  another  locality,  and  yet 
no  differences  can  be  seen  to  result  from  their  employ- 
ment. For  this  reason  the  proper  study  of  any  water, 
from  this  point  of  view,  means  the  establishment  of  what 
may  be  termed  its  normal  proportion  of  bacteria,  as  well 
as  a  study  of  the  organisms  most  commonly  present.  For. 
this  purpose  experiments  covering  a  long  period  of  time, 
made  at  short  intervals,  must  be  conducted,  and  from 
these  observations  the  means  for  that  water  at  the  dif- 
ferent seasons  of  the  year  calculated.  Marked  devia- 
tions from  these  means,  either  in  quantity  or  quality  of 
the  bacteria  present,  are  the  only  comparisons  that  are  of 
any  value. 

A  sudden  variation  from  the  normal  mean  in  the 
number  of  bacteria  in  any  water  calls  at  once  for  a 


186  BACTERIOLOGY. 

quantitative  chemical  analysis  as  well  as  a  thorough 
inspection  of  the  supply ;  at  the  same  time  the  character 
of  the  organisms  should  be  subjected  to  most  careful 
study. 

BACTERIOLOGICAL  AIR  ANALYSIS. — Quite  a  num- 
ber of  methods  for  the  bacteriological  study  of  the  air 
exist. 

In  the  main  they  consist  either  of  allowing  air  to  pass 
over  solid  nutrient  media  (Koch,  Hesse)  and  observing 
the  colonies  which  develop  upon  the  media,  or  of  filtering 
the  bacteria  from  the  air  by  means  of  porous  and  liquid 
substances,  and  studying  the  organisms  thus  obtained. 
(Miguel,  Petri,  Strauss,  Wiirz,  Sedgwick.) 

The  former  methods  have  given  place  almost  entirely 
to  the  latter  for  reasons  of  greater  exactness  possessed 
by  the  latter. 

In  some  of  the  methods  which  provide  for  the  filtra- 
tion of  bacteria  from  the  air  by  means  of  liquid  sub- 
stances, a  measured  volume  of  air  is  aspirated  through 
liquefied  gelatin;  this  is  then  rolled  into  an  Esmarch 
tube,  and  the  number  of  colonies  counted,  just  as  was 
done  in  the  water  analysis.  This  is  the  simplest  pro- 
cedure. An  objection  raised  against  it  is  that  organisms 
may  be  lost,  and  not  come  into  the  calculation,  by  pass- 
ing through  the  medium  in  the  centre  of  an  air-bubble 
without  being  arrested  by  the  fluid,  an  objection  that 
appears  more  of  speculative  than  of  real  value. 

The  methods  of  filtration  through  porous  substances 
appear,  on  the  whole,  to  give  the  best  results.  Petri 
recommends  the  aspiration  of  a  measured  volume  of  air 
through  glass  tubes  into  which  sterilized  sand  is  packed. 
(Fig.  30.)  When  the  aspiration  is  finished  the  sand 
is  mixed  with  liquefied  gelatin,  plates  are  made,  and 


SEDGWICK'S  METHOD.  187 

the  number  of  developing  colonies  counted,  the  results 
giving  the  number  of  organisms  contained  in  the  volume 
of  air  aspirated  through  the  sand. 

FIG.  30. 


The  tube  packed  with  sand  is  seen  at  the  point  a. 

The  main  objection  to  this  method  is  the  possibility 
of  mistaking  a  sand  granule  for  a  colony.  This  objec- 
tion has  been  overcome  by  Sedgwick,  who  employs 
granuated  sugar  instead  of  the  sand  ;  this  when  brought 
into  the  liquefied  gelatin  dissolves,  and  no  such  error 
as  that  possible  in  the  Petri  method  can  be  made. 

SEDGWICK'S  METHOD. — On  the  whole,  the  method 
proposed  by  Sedgwick  gives  such  uniform  results  that 
it  is  to  be  recommended  above  the  others.  It  is  as  fol- 
lows : 

The  apparatus  employed  consists  essentially  of  three 
parts : 

(1)  A  glass  tube  of  a  special  form  to  which  the  name 
aerobioscope  has  been  given. 


188  BACTERIOLOGY. 

(2)  A  stout  copper  cylinder  of  about  sixteen  litres 
capacity,  provided  with  a  vacuum-gauge. 

(8)  An  air-pump. 

The  aerobioscope  (Fig.  31)  is  about  35  cm.  in  its  en- 
tire length  ;  it  is  15  cm.  long  and  4.5  cm.  in  diameter  at 
its  expanded  part ;  one  end  of  the  expanded  part  is  nar- 
rowed down  to  a  neck  2.5  cm.  in  diameter  and  2.5  cm. 
long.  To  the  other  end  is  fused  a  glass  tube  15  cm. 
long  and  0.5  cm.  inside  diameter,  in  which  is  to  be 
placed  the  filtering  material. 


FIG.  31. 


Upon  this  narrow  tube,  5  cm.  from  the  lower  end,  a 
mark  is  made  with  a  file,  and  up  to  this  mark  a  small 
roll  of  brass- wire  gauze  (a)  is  inserted  ;  this  serves  as  a 
stop  for  the  filtering  material  which*  is  to  be  placed  over 
it.  Beneath  the  gauze  at  (6),  and  also  at  the  large  end 
(c),  the  apparatus  is  plugged  with  cotton.  When  thor- 
oughly cleaned,  dried,  and  plugged,  the  apparatus  is  to 
be  sterilized  in  the  hot-air  sterilizer.  "When  cool,  the 
cotton  plug  is  removed  from  the  large  end  (c),  and 
sterilized  No.  50  granulated  sugar  is  poured  in  until 
it  just  fills  the  10  cm.  (d)  of  the  narrow  tube  above 
the  wire  gauze.  This  column  of  sugar  is  the  filtering 
material  employed  to  engage  and  retain  the  microorgan- 
isms. After  pouring  in  the  sugar,  the  cotton-wool  plug 
is  replaced,  and  the  tube  is  again  sterilized  at  120°  C. 
for  several  hours. 


TAKING    THE    AIR    SAMPLE.  189 

Taking  the  air  sample.  In  order  to  measure  the 
amount  of  air  used,  the  value  of  each  degree  on  the 
vacuum-gauge  is  determined  in  terms  of  air  by  means 
of  an  air-meter,  or  by  calculation  from  the  known 
capacity  of  the  cylinder.  This  fact  ascertained,  the 
negative  pressure  indicated  by  the  needle  on  exhausting 
the  cylinder  shows  the  volume  of  air  which  must  pass 
into  it  in  order  to  fill  the  vacuum.  By  means  of  the 
air-pump  one  exhausts  the  cylinder  until  the  needle 
reaches  the  mark  corresponding  to  the  amount  of  air 
required. 

A  sterilized  aerobioscope  is  now  to  be  fixed  in  the  up- 
right position  and  its  small  end  connected  by  a  rubber 
tube  with  a  stop-cock  on  the  cylinder.  The  cotton  plug 
is  then  removed  from  the  upper  end  of  the  aerobioscope, 
and  the  desired  amount  of  air  is  aspirated  through  the 
sugar.  The  organisms  will  be  held  back  by  the  sugar. 
During  manipulation  the  cotton  plug  is  to  be  protected 
from  contamination  with  germs  from  without. 

When  the  required  amount  of  air  has  been  aspirated 
through  the  sugar  the  cotton  plug  is  replaced,  and  by 
gently  tapping  the  aerobioscope  while  held  in  an  almost 
horizontal  position,  the  sugar,  and  with  it  the  bacteria, 
are  brought  into  the  large  part  (e)  of  the  apparatus. 
When  all  the  sugar  is  thus  shaken  down  into  this  part 
of  the  apparatus,  about  20  c.c.  of  liquefied  sterilized 
gelatin  is  poured  in  through  the  opening  at  the  end 
c,  the  sugar  dissolves,  and  the  whole  is  then  rolled  on 
ice,  just  as  is  done  in  the  preparation  of  an  ordinary 
Es  march  tube. 

The  gelatin  is  most  easily  poured  into  the  aerobioscope 
by  the  use  of  a  small,  sterilized  cylindrical  funnel  (Fig. 

9* 


190  BACTERIOLOGY. 

32),  the  stem  of  which  is  bent  to  an  angle  of  about  110° 
with  the  long  axis  of  the  body. 

The  larger  part  of  the  aerobioscope  is  divided  into 
squares  to  facilitate  the  counting  of  the  colonies.  By 
the  employment  of  this  apparatus  one  can  make  these 
analyses  at  any  place,  and  can,  without  fear  of  con- 
tamination, carry  the  tubes  to  the  laboratory,  where  the 
cultivation  part  of  the  work  may  be  done. 

FIG.  32. 


Aside  from  this  advantage,  the  use  of  a  vacuum- 
cylinder  permits  a  known  volume  of  air  to  be  aspirated 
with  great  ease,  and  its  rate  of  flow  through  the  filter 
regulated  to  a  nicety. 

The  filter  being  soluble,  only  the  insoluble  bacteria 
are  left  imbedded  in  the  gelatin. 

For  general  use  this  method  is  to  be  preferred  to  the 
others  that  have  been  mentioned. 


BACTERIOLOGICAL    STUDY    OF    SOIL.       191 

BACTERIOLOGICAL  STUDY  OF  THE  SOIL. — Bacterio- 
logical study  of  the  soil  may  be  made  by  either  break- 
ing up  small  particles  of  earth  in  liquefied  media  and 
making  plates  directly  from  this,  or  by  what  is  per- 
haps a  better  method,  as  it  gets  rid  of  insoluble  parti- 
cles which  may  give  rise  to  errors  :  breaking  up  the  soil 
for  investigation  in  sterilized  water  and  then  making 
plates  from  the  water. 

It  "must  be  borne  in  mind  that  many  of  the  ground 
organisms  belong  to  the  anaerobic  group,  so  that  in  these 
studies  this  point  should  be  remembered  and  the  methods 
for  the  cultivation  of  these  organisms  practised  in  con- 
nection with  the  ordinary  methods. 


CHAPTER  XIX. 

Inoculation  experiments  with  sputum — Sputum  septicaemia — 
Septicaemia  resulting  from  the  presence  of  the  micrococcus  tetra 
genus  in  the  tissues — Tuberculosis. 

OBTAIN  from  a  tuberculous  patient  a  sample  of  fresh 
sputum — that  of  the  morning  is  preferable.  Spread  it 
out  in  a  thin  layer  upon  a  black  glass  plate  and  select 
one  of  the  small,  white,  cheesy  masses  or  dense  mucous 
clumps  that  will  be  seen  scattered  through  the  sputum. 
With  a  pointed  forceps  smear  it  carefully  upon  two 
or  three  thin  cover-slips,  dry  and  fix  them  in  the 
way  given  for  ordinary  cover-slip  preparations.  Stain 
one  in  the  ordinary  way  with  Loffler's  alkaline  methy- 
lene-blue  solution,  the  other  by  the  Gram  method,  the 
third  after  the  method  given  for  tubercle  bacilli  in  fluids 
or  sputum. 

In  that  stained  by  Loffler's  method — slip  No.  1 — will 
be  seen  a  great  variety  of  organisms — round  cells,  ovals, 
short  and  long  rods,  perhaps  spiral  forms.  But  not  in- 
frequently will  be  seen  diplococci,  having  more  or  less 
of  a  lancet  shape ;  they  will  be  joined  together  by  their 
broad  ends,  the  points  of  the  lancet  being  away  from  the 
point  of  juncture  of  the  two  cells.  There  may  also  be 
seen  masses  of  cocci  which  are  conspicuous  for  their  ar- 
rangement into  groups  of  fours,  the  adjacent  surfaces 
being  somewhat  flattened.  They  are  not  sarcinae,  as  one 
can  see  by  the  absence  of  the  division  in  the  third  direc- 
tion— they  divide  only  in  two  directions. 


INOCULATION    WITH    SPUTUM.  193 

In  the  slip  stained  by  the  Gram  method  the  same 
groups  of  the  cocci  which  grow  as  threes  and  fours  will 
be  seen,  but  our  lancet-shaped  diplococci  will  now  pre- 
sent an  altered  appearance — there  can  now  be  detected  a 
capsule  surrounding  them.  This  capsule  is  very  deli- 
cate in  structure,  and  though  a  frequent  accompaniment 
is  not  constant  It  can  sometimes  be  demonstrated  by 
the  ordinary  methods  of  staining,  though  the  method  of 
Gram  is  most  satisfactory. 

In  the  third  slip,  which  has  been  stained  by  the 
method  given  for  tubercle  bacilli  in  sputum,  if  de- 
colorization  has  been  properly  conducted  and  no  con- 
trast stain  has  been  employed,  the  field  will  be  color- 
less or  of  only  a  very  pale  rose  color.  None  of  the 
numerous  organisms  seen  in  the  first  slip  can  now  be 
detected,  but  instead  there  will  be  seen  scattered  through 
the  field  very  delicate  stained  rods,  which  present,  in 
most  instances,  a  conspicuous  beaded  arrangement  of 
their  protoplasm — that  is,  the  staining  is  not  homoge- 
neous, but  at  tolerably  regular  intervals  along  each  rod 
there  is  seen  alternating  intervals  of  light  and  color^ 
These  rods  may  be  found  singly,  in  groups  of  twos  or 
threes,  or  sometimes  in  clumps  consisting  of  large  num- 
bers. When  in  twos  or  threes  it  is  not  uncommon  to 
find  them  describing  an  X  or  a  V  in  their  mode  of  ar- 
rangement, or  again  they  will  be  seen  lying  paralkl  the 
one  to  the  other. 

If  contrast  stains  are  used,  these  rods  will  be  detected 
and  recognized  by  their  retaining  the  original  color  with 
which  they  have  been  stained,  whereas  all  other  bac- 
teria in  the  preparation,  as  well  as  the  tissue-cells  which 
are  in  the  sputum,  will  take  up  the  contrast  color. 

These  delicate  beaded  rods  are  the  tubercle  bacilli. 


194  BACTERIOLOGY. 

The  lancet-shaped  diplococci  with  the  capsule  are  the 
diplococci  of  sputum  septicaemia. 

The  cocci  grouped  in  fours  are  the  micrococcus  tetra- 
genus. 

INOCULATION  EXPERIMENT. — Inoculate  into  the 
subcutaneous  tissues  of  a  guinea-pig  one  of  the  small 
white  caseous  masses  similar  to  that  which  has  been 
examined  microscopically.  If  death  ensues  it  will  be 
the  result  of  one  of  the  three  following  forms  of 
infection  : 

a.  Septicaemia1  resulting  from  the  introduction  into 
the  tissues  of  an  organism  frequently  present  in  the 
sputum.     It  exists   under  the  various   names :   micro- 
coccus  of  sputum  septicaemia ;  diplococcus  pneumouiae ; 
pneumococcus   of  Frankel ;   diplococcus   lanceolatus — 
lancet-formed  diplococcus ;  meningococcus ;  streptococcus 
lanceolatus  Pasteuri. 

b.  A  form  of  septicaemia  resulting  from  the  invasion 
of  the  tissues  by  an  organism  frequently  seen  in  the 
sputum  of  tuberculous  subjects.     It  is  characterized  by 
its  tendency  to  divide  into  fours.     It  is  the  micrococcus 
tetragenus. 

c.  General  or  local  tuberculosis. 

a.    SPUTUM    SEPTIC^MIA. 

If  at  the  end  of  twenty-four  to  thirty-six  hours  the 
animal  is  found  dead,  we  may  safely  suspect  that  the 
result  was  produced  by  the  introduction  into  the  tissues 
of  the  organism  of  sputum  septicaemia  above  mentioned, 
which  is  not  uncommonly  found  in  the  mouth  of  healthy 
individuals  as  well  as  in  other  conditions. 

1  Septicaemia  is  that  form  of  infection  in  which  the  blood  is  the 
chief  field  of  activity  of  the  organisms. 


SPUTUM    SEPTIC^MIA.  195 

Inspection  reveals  nothing  abnormal  at  the  seat  of 
inoculation,  except  when  death  is  postponed  for  a  longer 
time,  when  some  oedema  may  be  present.  At  autopsy 
the  most  conspicuous  naked-eye  change  will  be  en- 
largement of  the  spleen.  Frequently  there  is  a  limited 
fibrinous  exudation  over  portions  of  the  peritoneum. 

Except  in  the  exudations,  the  organisms  are  found 
only  in  the  lumen  of  the  bloodvessels,  where  they  are 
usually  present  in  enormous  numbers. 

In  the  blood  they  are  always  free  and  are  not  found 
in  the  body  of  leucocytes. 

In  stained  preparations  from  the  blood  and  exudates 
a  capsule  is  not  unfrequently  seen  surrounding  the  organ- 
isms. This,  however,  is  not  constant. 

If  a  drop  of  blood  from  this  animal  be  introduced 
into  the  tissues  of  a  second  animal  (mouse,  rabbit,  or 
guinea-pig),  identically  the  same  conditions  will  be. re- 
produced. 

If  the  organism  be  isolated  from  the  blood  of  the 
animal  in  pure  culture,  and  a  portion  of  this  culture  be 
introduced  into  the  tissues  of  a  susceptible  animal,  again 
we  shall  see  the  same  pathological  picture. 

It  must  be  remembered,  however,  that  this  organism 
when  cultivated  for  a  time  on  artificial  media  rapidly 
loses  its  virulent  properties.  If,  therefore,  failure  to  re- 
produce the  disease  after  inoculation  from  old  cultures 
should  occur,  it  is,  in  all  probability,  due  to  a  disappear- 
ance of  virulence  from  the  organism. 

This  organism  was  discovered  by  Stern  berg  in  1880. 
It  was  subsequently  described  by  A.  Frankel  as  the 
etiological  factor  in  the  production  of  acute  fibrinous 
pneumonia. 

It  is  not  uncommonly  present  in  the  saliva  of  healthy 


196  BACTEKIOLOGY. 

individuals,  having  been  found  by  Sternberg  in  the  oral 
cavity  of  about  20  per  cent,  of  healthy  persons  exam- 
ined by  him.  It  is  constantly  to  be  detected  in  the 
rusty  sputum  of  patients  suffering  from  acute  fibrinous 
pneumonia.  Its  presence  has  been  detected  in  the 
middle  ear,  in  the  pericardial  sac,  in  the  pleura,  in  the 
serous  cavities  of  the  brain,  and  indeed  it  may  probably 
penetrate  from  its  primary  seat  in  the  mouth  to  almost 
any  of  the  more  distant  organs. 

The  organism  is  commonly  found  as  a  diplococcus, 
though  here  and  there  short  chains  of  four  to  six  indi- 
viduals joined  together  may  be  detected.  The  mor- 
phology of  the  individual  cells  is  more  or  less  oval,  or 
more  strictly  speaking,  lancet-shaped,  for  at  one  end 
there  is  commonly  a  pointed  appearance.  When  joined 
in  pairs  the  junction  is  always  between  the  broad  ends 
of  the  ovals,  never  between  the  pointed  extremities. 

In  preparations  directly  from  the  sputum  or  from  the 
blood  of  animals,  a  delicate  capsule  may  frequently  be 
seen  surrounding  them.  This  occurs  only  in  prepara- 
tions directly  from  the  body  and  is  not  seen  in  pre- 
parations from  cultures. 

This  organism  grows  under  artificial  conditions  very 
slowly,  and  frequently  not  at  all. 

When  successfully  grown  upon  the  different  media  it 
presents  somewhat  the  following  appearance  : 

On  gelatin  it  grows  very  slowly,  probably  owing  in 
part  to  the  low  temperature  at  which  gelatin  cultures 
must  be  kept.  If  development  occurs  it  appears  as 
very  small  whitish  or  blue-white  points  on  the  plates. 
These  very  small  colonies  are  round,  finely  granular, 
sharply  circumscribed,  and  slightly  elevated  above  the 


SPUTUM    SEPTICAEMIA.  197 

surface  of  the  gelatin.  The  growth  is  very  slow  and  no 
liquefaction  of  the  gelatin  occurs. 

If  grown  in  slant  or  stab  cultures,  the  surface  devel- 
opment is  very  limited;  along  the  needle  track  very 
small,  whitish  granules  appear.  This  organism  is  con- 
spicuous for  the  rapidity  with  which  it  loses  its  patho- 
genic properties  and  the  fact  that  after  a  very  few  gen- 
erations it  can  no  longer  be  caused  to  grow. 

On  agar-agar  the  colonies  are  almost  transparent ;  they 
are  more  or  less  glistening  and  very  delicate  in  their 
structure. 

On  blood-serum  the  growth  is  more  marked,  though 
still  extremely  feeble.  Here  it  appears  as  a  very  deli- 
cate film,  consisting  of  fine  points  growing  closely  side 
by  side. 

A  growth  on  potato  has  not  been  observed. 

The  organism  is  not  motile. 

It  grows  best  at  a  temperature  between  35°  C.-380  C. 

Under  24°  C.  no  growth  has  been  observed,  and  from 
42°  C.  on,  the  development  is  checked. 

Under  most  favorable  conditions  the  growth  is  very 
slow.  It  grows  as  well  without  as  with  oxygen.  It  is, 
therefore,  one  of  the  facultative  anaerobic  forms. 

The  most  successful  efforts  at  the  cultivation  of  this 
organism  are  those  seen  when  the  agar-gelatin  mixture 
of  Guarniari  is  employed.  (See  this  medium.) 

It  may  be  stained  with  the  ordinary  aniline  staining 
reagents.  For  demonstration  of  the  capsule  the  method 
of  Gram  gives  the  best  results.  (See  Stainings.) 

b.   SEPTICAEMIA    OF   THE   MICROCOCCUS  TETRAGENUS. 

Should  the  death  of  the  animal  not  occur  within 
the  first  twenty-eight  to  thirty  hours  after  inoculation, 


198  BACTERIOLOGY. 

but  be  postponed  until  between  the  fourth  and  the  eighth 
day,  it  may  occur  as  a  result  of  invasion  of  the  tissues 
by  the  organism  now  to  be  described,  viz.,  the  micro- 
coccus  tetragenus. 

This  organism  was  discovered  by  Gaff ky  and  was 
subsequently  described  by  Koch  in  the  account  of  his 
experiments  upon  tuberculosis.  It  is  often  present  in 
the  saliva  of  healthy  individuals  and  is  commonly 
present  in  the  sputum  of  tuberculous  patients.  Koch 
found  it  very  frequently  in  the  lung  cavities  of 
phthisical  patients.  It,  however,  plays  no  part  in  the 
etiology  of  tuberculosis. 

It  is  a  small  round  coccus  of  about  1  p  transverse 
diameter.  It  is  seen  as  single  cells,  joined  in  pairs  and 
in  threes,  but  its  most  conspicuous  grouping  is  in  fours, 
from  which  arrangement  it  takes  its  name.  In  prepa- 
rations made  from  cultures  of  this  organism  it  is  not 
rare  to  find  here  and  there  single  bodies  which  are  much 
larger  than  the  other  individuals  in  the  field.  Close  in- 
spection reveals  these  bodies  to  be  cells  in  the  initial 
stage  of  division  into  twos  and  fours.  A  peculiarity  of 
this  organism  is  that  the  cells  are  seen  to  be  bound 
together  by  a  transparent  gelatinous  substance. 

When  cultivated  artificially  it  grows  very  slowly. 

Upon  gelatin  plates  the  colonies  appear  as  round, 
sharply  circumscribed,  punctiform  masses  which  are 
slightly  elevated  above  the  surface  of  the  surrounding 
medium.  Under  a  low  magnifying  power  they  are  seen 
to  be  slightly  granular  and  present  a  more  or  less  glassy 
lustre. 

The  colonies  increase  but  little  in  size  after  the  third 
or  fourth  day.  If  cultivated  as  stab  cultures  in  gelatin 
there  appears  upon  the  surface  at  the  point  of  inocula- 


SEPTIC^MIA    OF    M.  TETRAGENUS.         199 

tion  a  circumscribed  white  point,  slightly  elevated  above 
the  surface  and  limited  to  the  immediate  neighborhood 
of  the  point  of  inoculation.  Down  the  needle-track 
the  growth  is  not  continuous,  but  appears  in  isolated, 
round,  dense  white  clumps  or  beads,  which  do  not 
develop  beyond  the  size  of  very  small  points. 
It  does  not  liquefy  gelatin. 

Upon  plates  of  nutrient  agar-agar  the  colonies  appear 
as  small,  almost  transparent,  round  points,  which  have 
about  the  same  color  as  a  drop  of  egg-albumin;  they  are 
very  slightly  opaque.  They  are  moist  and  glistening. 
They  rarely  develop  to  an  extent  exceeding  1  to  2  mm. 
in  diameter. 

Upon  agar-agar  as  stab  or  slant  cultures,  the  surface 
growth  has  more  or  less  of  a  mucoid  appearance.  It  is 
moist,  glistening,  and  irregularly  outlined.  The  outline 
of  the  growth  depends  upon  the  moisture  of  the  agar. 
It  is  slightly  elevated  above  the  surface  of  the  medium. 
In  contradistinction  to  the  gelatin  stab- cultures,  the 
growth  is  continuous  along  the  track  of  the  needle  in 
the  stab  cultures  upon  agar-agar. 

The  growth  on  potato  is  a  thick,  irregular,  slimy- 
looking  patch. 

The  presence  of  the  transparent  gelatinous  substance 
which  is  seen  to  surround  these  organisms  renders  them 
coherent,  so  that  efforts  to  take  up  a  portion  of  a  colony 
from  the  agar-agar  or  potato  cultures  results  usually  in 
drawing  out  fine  silky  threads  consisting  of  organisms 
imbedded  in  this  gelatinous  material. 

The  organism  grows  best  at  from  35°  C.  to  38°  C., 
but  can  be  cultivated  at  the  ordinary  room  temperature 
—about  20°  C. 

The  growth  under  all  conditions  is  not  rapid. 


200  BACTERIOLOGY. 

It  grows  both  in  the  presence  of  and  without  oxygen. 

It  is  not  motile. 

It  stains  readily  with  all  the  ordinary  aniline  dyes. 
In  tissues  its  presence  is  readily  demonstrated  by  the 
staining  method  of  Gram. 

The  grouping  into  fours  is  particularly  well  seen  in 
sections  from  the  organs  of  animals  dead  of  this  form 
of  septicaemia. 

In  such  sections  the  organisms  will  always  be  found 
within  the  capillaries. 

To  the  naked  eye  no  alteration  can  be  seen  in  the 
organs  of  animals  which  have  died  as  a  result  of  inocu- 
lation with  the  micrococcus  tetragenus ;  but  micro- 
scopic examination  of  cover-slip  preparations  from  the 
blood  and  viscera  reveals  the  presence  of  the  organisms 
throughout  the  body — especially  is  this  true  of  prepara- 
tions from  the  spleen.  White  mice  and  guinea-pigs  are 
susceptible  to  the  disease.  Gray  mice,  dogs,  and  rabbits 
are  not  susceptible  to  this  form  of  septicaemia.  Subse- 
quent inoculation  of  healthy  animals  with  a  drop  of 
blood,  a  bit  of  tissue,  or  a  portion  of  a  pure  culture  of 
this  organism  from  the  body  of  an  animal  dead  of  the 
disease,  results  in  a  reproduction  of  the  conditions  found 
in  the  dead  animal  from  which  the  tissues  or  cultures 
were  obtained. 

It  sometimes  occurs  that  in  guinea-pigs  which  have 
been  inoculated  with  this  organism,  there  results  local 
pus-formations  instead  of  a  general  septicaemia.  The 
organisms  will  then  be  found  in  the  pus-cavity. 


CHAPTER  XX. 

Tuberculosis — Microscopic  appearance  of  miliary  tubercles— En- 
capsulation of  tuberculous  foci — Diffuse  caseation — Cavity-formation 
— Primary  infection— Modes  of  infection — Location  of  the  bacilli 
in  the  tissues — Staining  peculiarities. 

SHOULD  the  animal  succumb  to  neither  of  the  septic 
processes  just  described,  then  its  death  from  tuberculosis 
may  be  reasonably  expected. 

When  this  process  is  in  progress,  alterations  in  the 
lymphatic  glands  nearest  the  seat  of  inoculation  may  be 
detected  by  the  touch  in  from  two  to  four  weeks.  They 
will  then  be  found  to  be  enlarged.  Though  not  constant, 
tumefaction  and  subsequent  ulceration  at  the  point  of 
inoculation  may  sometimes  be  observed.  Progressive 
emaciation,  loss  of  appetite,  and  difficulty  in  respiration 
point  to  the  existence  of  the  tubercular  process.  Death 
ensues  in  from  four  to  eight  weeks  after  inoculation. 
At  autopsy  either  general  or  local  tuberculosis  may  be 
found.  The  expressions  of  the  tubercular  process  are 
so  manifold  and  in  different  animals  differ  so  widely  the 
one  from  the  other,  that  no  rigid  law  as  to  what  will  be 
found  at  autopsy  can  a  priori  be  laid  down. 

The  guinea-pig,  which  is  best  suited  for  this  experi- 
ment, because  of  the  greater  regularity  of  its  suscepti- 
bility to  the  disease  over  that  of  other  animals  usually 
found  in  the  laboratory,  presents,  in  the  main,  changes 
which  are  characterized  by  a  condition  of  coagulation, 
necrosis,  and  caseation.  This  is  particularly  the  case 
when  the  infection  is  general,  i.  e.,  when  the  process  is 


202  BACTERIOLOGY. 

of  the  acute  miliary  type.  This  pathological-anatomical 
alteration  is  best  seen  in  the  tissue  of  the  liver  and 
spleen  of  these  animals,  where  the  condition  is  most 
pronounced. 

In  general,  the  tubercular  lesions  can  be  divided  into 
those  of  strictly  focal  character — the  miliary  and  the 
conglomerate  tubercles,  and  those  which  are  more  diffuse 
in  their  nature.  The  latter  lesions,  although  of  the  same 
fundamental  nature  as  the  miliary  tubercles,  are  much 
greater  in  extent  and  not  so  sharply  circumscribed. 

These  latter  lesions  play  a  greater  role  in  the  path- 
ology of  the  disease  than  do  the  miliary  nodules,  although 
it  is  to  the  presence  of  the  latter  that  the  disease  owes 
its  name. 

At  autopsy  the  pathological  manifestations  of  the 
disease  are  not  infrequently  confined  to  the  seat  of 
inoculation  and  the  neighboring  lymphatic  glands. 
These  tissues  will  then  present  all  the  characteristics 
of  the  tuberculous  process  in  the  stage  of  cheesy  de- 
generation. When  the  disease  is  general  the  degree 
of  its  extension  varies.  Sometimes  the  small  gray 
nodules — the  miliary  tubercles — are  only  to  be  seen  with 
the  naked  eye  in  the  tissues  of  the  liver  and  spleen. 
Again,  they  may  invade  the  lungs,  and  commonly 
they  are  distributed  over  the  serous  membranes  of 
the  intestines,  the  lungs,  the  heart,  and  the  brain. 
These  simple  gray  nodules,  as  seen  by  the  naked  eye, 
vary  in  size  from  that  of  a  pin-point  to  that  of  a  hemp- 
seed,  and  as  a  rule  are,  in  this  stage,  the  result  of  the 
fusion  between  two  or  more  smaller  miliary  foci.  Though 
the  two  terms,  "  miliary  "  and  u  conglomerate,"  exist 
for  the  description  of  the  macroscopic  appearance  of 
these  nodules,  yet  it  is  very  rare  that  any  condition 


MILIARY    TUBERCLES.  203 

other  than  that  due  to  the  fusion  together  of  several  of 
these  minute  foci  can  be  detected  by  the  naked  eye. 

The  miliary  tubercles  are  of  a  pale  gray  color,  with  a 
white  centre,  are  slightly  elevated  above  the  surface  of 
the  tissue  in  which  they  exist,  and,  as  stated,  vary  con- 
siderably in  dimensions,  usually  appearing  as  points 
which  range  in  size  from  that  of  a  pin-point  to  that  of  a 
pin-head.  They  are  not  only  located  upon  the  surface  of 
the  organs,  but  are  distributed  through  the  depths  of 
the  tissues.  To  the  touch  they  sometimes  present  nothing 
characteristic,  but  may  frequently,  when  closely  packed 
together  in  large  numbers,  give  a  mealy  or  sandy  sensa- 
tion to  the  fingers.  Stained  sections  of  these  miliary 
tubercles  present  an  entirely  characteristic  appearance, 
and  the  disease  may  be  diagnosticated  by  these  histologi- 
cal  changes  alone,  though  the  crucial  test  in  the.diagnosis 
is  the  finding  of  tubercle  bacilli  in  these  nodules. 

MICROSCOPIC  APPEARANCE  OF  MILIARY  TUBER- 
CLES.— The  simple  miliary  tubercles  under  the  low  mag- 
nifying power  of  the  microscope  present  somewhat  the  fol- 
lowing appearance :  There  is  a  central  pale  area,  evidently 
composed  of  necrotic  tissue  because  of  its  incapacity  for 
taking  up  the  staining  employed.  Scattered  here  and 
there  through  this  necrotic  area  may  be  seen  granular 
masses  irregular  in  size  and  shape.  They  take  up  the 
stains  employed  and  are  evidently  the  fragments  of  cell- 
nuclei  in  the  course  of  destruction.  Through  the 
necrotic  area  may  here  and  there  be  seen  irregular  lines, 
bands,  or  ridges,  the  remains  of  tissues  not  yet  com- 
pletely destroyed  by  the  necrotic  process.  Around 
the  periphery  of  this  area  may  sometimes  be  noticed 
large  mu It i- nucleated  cells,  the  nuclei  of  which  are 
arranged  about  the  periphery  of  the  cell  or  grouped 


204  BACTERIOLOGY. 

irregularly  at  its  poles.  The  arrangement  of  these 
nuclei  appears  in  the  sections  sometimes  as  ovals,  again 
they  are  somewhat  crescentic  in  their  grouping.  In  the 
tubercles  from  the  human  subject  these  large  "giant- 
cells,"  as  they  are  called,  are  quite  common.  They  are 
much  less  frequent  in  the  tubercular  tissues  from  the 
lower  animals. 

Round  about  this  central  focus  of  necrosis  is  seen  a 
more  or  less  broad  zone  of  closely  packed  small  round 
and  oval  bodies  which  stain  readily  but  not  homogene- 
ously. They  vary  in  size  and  shape,  and  are  seen  to  be 
imbedded  in  a  delicate  network  of  fibrous-looking  tissue. 

This  fibrous-looking  network  in  which  these  bodies 
lie,  and  which  is  a  common  accompaniment  of  giant-cell 
formation,  is  in  part  composed  of  fibrin,  but  is  in  the 
main  most  probably  the  remains  of  the  interstitial 
fibrous  tissue  of  the  part.  This  zone  of  which  we  are 
speaking,  is  the  zone  of  so-called  "  granulation  tissue," 
and  consists  of  leucocytes,  granulation  cells,  and  the 
fibrous  remains  of  the  part ;  the  irregularly  oval,  granu- 
lar bodies  which  take  up  the  staining  are  the  nuclei  of 
these  cells.  The  zone  of  granulation  tissue  surrounds 
the  whole  of  the  tubercular  process,  and  at  its  periphery 
fades  gradually  into  the  healthy  surrounding  tissue  or 
fuses  with  a  similar  zone  surrounding  another  tuber- 
cular focus.  This  may  be  taken  as  the  description  of  a 
typical  miliary  tubercle. 

DIFFUSE  CASSATION. — The  diffuse  caseation,  as  said, 
plays  a  more  important  role  in  the  tuberculous  lesion, 
both  in  the  human  and  experimental  forms,  than  does 
the  formation  of  miliary  tubercles.  In  this  a  large  area 
of  tissue  undergoes  the  same  process  of  necrosis  and 
caseatiou  as  the  centre  of  the  miliary  tubercle.  In  some 


DIFFUSE    CASEATION.  205 

tissues  it  is  more  marked  than  in  others.  These  tissues 
are  the  lungs  and  the  lymph-glands.  In  rabbits,  par- 
ticularly, all  the  changes  in  the  lung  frequently  come 
under  this  head.  When  this  is  the  case  solid  masses  are 
found,  sometimes  as  large  as  a  pea,  or  involving  even 
an  entire  lobe  or  the  whole  lung  in  some  cases.  They 
are  of  a  whitish-yellow,  opaque  color,  and  on  section 
are  peculiarly  dry  and  hard.  Entire  lymphatic  glands 
may  be  changed  in  this  way.  The  conditions  for  this 
caseation  of  the  tissues  are  probably  given  when  a  large 
number  of  tubercle  bacilli  enter  the  tissue  simultaneously 
and  a  wide  area  is  involved,  instead  of  the  small  centre 
of  the  miliary  tubercle.  Necrosis  is  so  rapid  that  time 
is  not  given  for  those  reactive  changes  to  take  place  in 
the  tissues  which  result  in  the  formation  of  the  outer 
zone  of  the  miliary  tubercle.  In  other  instances  the  en- 
tire caseous  area  is  surrounded  by  a  zone  similar  to  that 
around  the  caseous  centre  of  the  miliary  tubercles.  It 
is  of  special  importance  to  recognize  the  connection  be- 
tween this  diffuse  caseation  of  the  tissue  and  the  tubercle 
bacilli,  because  until  its  nature  was  accurately  deter- 
mined the  caseous  pneumonia  of  the  lungs  formed  the 
chief  obstacle  which  many  had  in  recognizing  the  infec- 
tiousuess  of  tuberculosis. 

CAVITY-FORMATION. — The  production  of  cavities 
which  forms  such  a  prominent  feature  in  human  tuber- 
culosis, particularly  in  the  lungs,  is  due  to  the  softening  of 
the  necrotic  caseous  masses  or  of  aggregations  of  miliary 
tubercles.  The  material  softens  and  is  expelled,  and  a 
cavity  remains.  In  the  wall  of  this  cavity  the  tuber- 
culous changes  still  proceed,  both  as  a  diffuse  caseation 
and  formation  of  miliary  tubercles.  The  whole  cavity 
with  the  reactive  changes  in  the  tissues  of  its  walls  may 

10 


206  BACTERIOLOGY. 

be  considered  as  representing  a  single  tubercle,  its  wall 
forming  a  tissue  very  analogous  to  the  outer  zone  of 
the  single  tubercle,  the  cavity  itself  corresponding  to  the 
caseous  centre.  In  the  lower  auimals  cavity-formation 
of  this  sort  is  very  rare,  owing  to  the  greater  resistance 
of  the  caseous  tissue. 

In  the  contents  and  in  the  walls  of  tubercular  cavities 
bacteria  other  than  the  tubercle  bacilli  are  found.  It  is 
to  the  influence  of  some  of  these,  as  we  have  just  seen, 
that  diseases  other  than  tuberculosis  may  sometimes  be 
produced  by  the  inoculation  of  lower  auimals  with  the 
sputum  from  such  cases. 

ENCAPSULATION  OF  TUBERCULAR  Foci. — It  not  un- 
commonly occurs  that  round  about  a  necrotic  tubercular 
focus  there  is  formed  a  fibrous  capsule  which  may  com- 
pletely cut  off  the  diseased  from  the  healthy  tissue  sur- 
rounding it.  Or  a  tubercular  focus  may,  through  the 
resistance  of  the  tissue  in  which  it  is  located,  be  more  or 
less  completely  isolated.  In  this  condition  the  diseased 
foci  may  lie  dormant  for  a  long  time  and  give  no  evidence 
of  their  existence,  until  by  some  intercurrent  interfer- 
ence they  are  caused  to  break  through  their  envelopes. 
With  the  passage  of  the  bacilli  or  their  spores  from 
the  central  foci  into  the  vascular  or  lymphatic  circula- 
tion the  disease  may  then  become  general. 

It  is  to  some  such  accident  as  this  that  the  sudden 
appearance  of  general  tubercular  infection  in  subjects 
supposed  to  have  recovered  from  the  primary  local 
manifestations  may  often  be  attributed.  The  breaking- 
down  of  old  caseous  lymphatic  glands  is  a  common 
example  of  this  condition. 

PBIMARY  INFECTION. — The  primary  infection  occurs 
through  either  the  vascular  or  lymphatic  circulation* 


MODES    OF    TUBERCULAR    INFECTION.      207 

Through  these  channels  the  bacilli  gain  access  to  the 
tissues  and  become  lodged  in  the  finer  capillary  ramifi- 
cations or  in  the  more  minute  lymph- spaces.  Here 
they  find  conditions  favorable  to  their  development,  and 
in  the  course  of  this  process  produce  substances  of  a 
chemical  nature  which  act  directly  in  bringing  about  the 
death  of  the  tissues  in  their  immediate  neighborhood. 
This  tissue-death  is  probably  the  very  first  effect  of  the 
bacilli  in  the  body,  and  represents  the  necrotic  centre, 
which  can  always  be  seen  in  even  the  most  minute 
tubercles.  With  the  production  of  this  progressive 
necrosis — for  progressive  it  is,  as  it  continues  as  long 
as  the  bacilli  live  and  continue  to  produce  their  poison- 
ous products — there  is  in  addition  a  reactive  change  in 
the  surrounding  tissues  which  consists  in  the  formation 
of  the  granulation  zone  at  the  outer  margins  of  the 
dying  and  dead  tissue.  This  zone  consists  of  small, 
round  granulation-cells  and  of  leucocytes,  all  of  which 
are  seen  in  the  meshes  of  the  finer  fibrous  tissues  of  the 
part.  At  the  same  time  alterations  are  produced  in  the 
walls  of  the  vessels  going  to  the  part;  this  tends  to 
occlude  them,  and  thus  the  process  of  tissue-death  is 
favored  by  a  diminution  of  the  amount  of  nutrition 
brought  to  them.  These  changes  continue  until  eventu- 
ally the  life  processes  of  the  bacilli  are  checked,  or 
conglomerate  tubercles,  widespread  caseation,  or  cavity- 
formation  results. 

MODES  OF  INFECTION. — Experimentally,  tuberculosis 
may  be  produced  in  susceptible  animals  by  subcutaneous 
inoculation,  by  direct  injection  into  the  circulation,  by 
injection  into  the  peritoneal  cavity,  by  feeding  of  tuber- 
culous material,  and  by  the  introduction  of  the  bacilli 
into  the  air-passages. 


208  BACTERIOLOGY. 

In  the  human  subject  the  most  common  portals  of 
infection  are  doubtless  the  air- passages,  the  alimentary 
tract,  and  cutaneous  wounds.  When  introduced  sub- 
cutaneously  the  resulting  process  finds  its  most  pro- 
nounced expression  in  the  lymphatic  system.  The 
growing  bacilli  make  their  way  into  the  fine  lymphatic 
spaces  of  the  loose  cellular  tissue,  are  taken  up  in  the 
lymph  stream  and  deposited  in  the  neighboring  lymph- 
atic glands.  Here  they  may  remain,  and  give  rise  to  no 
alteration  further  than  that  seen  in  the  glands  themselves, 
or  they  may  pass  on  to  neighboring  glands  and  eventually 
be  disseminated  throughout  the  whole  lymphatic  system, 
ultimately  reaching  the  vascular  system. 

When  having  gained  access  to  the  bloodvessels,  the 
results  are  the  same  as  those  following  upon  intra- 
vascular  injection  of  the  bacilli :  general  tuberculosis 
quickly  follows,  with  the  most  conspicuous  production 
of  miliary  tubercles  in  the  lungs  and  kidneys,  less 
numerous  in  the  spleen,  liver,  and  bone  marrow. 

When  inhaled  into  the  lungs,  if  conditions  are  favor- 
able, multiplication  of  the  bacilli  quickly  follows.  With 
their  growth  they  are  mechanically  pressed  into  the  tis- 
sues of  the  lungs;  as  multiplication  continues  some 
are  transported  from  the  primary  seat  of  infection  to 
healthy  portions  of  the  lung  tissue,  there  to  give  rise  to 
a  further  production  of.  the  tubercular  process. 

In  the  same  way  infection  through  the  alimentary 
tract  is  in  the  main  due  to  the  mechanical  pressure. of 
the  bacilli  upon  the  walls  of  the  intestines.  Investiga- 
tion has  shown  that  lesions  of  the  intestinal  coats  are 
not  necessary  for  the  entrance  of  the  tubercle  bacilli 
from  the  intestines  into  the  body.  They  may  be  trans- 
ported from  the  intestinal  tract  into  the  lymphatics  in 


LOCATION    OF    BACILLI    IN    THE    TISSUES.       209 

the  same  way  that  the  fat  droplets  of  the  chyle  find 
entrance  into  the  lymphatic  circulation. 

The  evidence  produced  by  Cornet  points  to  the  lungs 
as  the  most  common  portals  of  natural  infection  for  the 
human  being.  Unlike  most  pathogenic  organisms,  the 
tubercle  bacillus  has  the  property  of  forming  spores 
within  the  tissues.  These  spores,  which  are  highly  re- 
sistant and  are  not  destroyed  by  drying,  are  thrown  off 
from  the  lungs  in  the  sputum  of  tuberculous  patients  in 
large  number,  and  unless  special  precautions  are  taken 
to  prevent  it  the  sputum  becomes  dried,  is  ground 
into  dust  and  sets  free  in  the  atmosphere  the  spores 
of  tubercle  bacilli  which  came  with  it  from  the  lungs. 
The  frequency  of  pulmonary  tuberculosis  points  to  this 
as  one  of  the  commonest  sources  of  infection. 

LOCATION  OF  THE  BACILLI  IN  THE  TISSUES. — The 
bacilli  will  be  found  to  be  most  numerous  in  those  tis- 
sues which  are  in  the  active  stage  of  the  process. 

In  the  very  initial  stage  of  the  disease  the  bacilli  will 
be  fewer  in  number  than  later.  .At  this  stage  only  here 
and  there  single  rods  may  be  found ;  later  they  will  be 
more  numerous,  and  finally,  when  the  process  has  ad- 
vanced to  a  stage  easily  recognizable  by  the  naked 
eye,  they  will  be  found  in  the  granulation  zones  in 
clumps  and  scattered  about  in  large  numbers. 

In  the  central  uecrotic  masses,  which  consist  of  cell 
detritus,  it  is  rare  that  the  organisms  can  be  demon- 
strated microscopically.  It  is  at  the  periphery  of  these 
areas  and  in  the  progressing  granular  zone  that  they  are 
most  frequently  to  be  seen. 

This  apparent  absence  of  the  bacilli  from  the  central 
necrotic  area  must  not  be  taken,  however,  as  evidence 
that  this  tissue  does  not  contain  them.  As  bacilli,  they 


210  BACTERIOLOGY. 

are  difficult  to  demonstrate  here  because  the  probabili- 
ties are  that  in  this  locality,  owing  to  conditions  unfa- 
vorable to  their  further  growth,  they  are  iu  the  spore 
stage,  a  stage  in  which  it  is  as  yet  impossible,  with  our 
present  methods  of  staining,  to  render  them  visible.  The 
fact  that  this  tissue  is  infective,  and  with  it  the  disease 
can  be  reproduced  in  susceptible  animals,  speaks  for  the 
accuracy  of  this  assumption.  A  conspicuous  example 
of  this  condition  is  seen  in  old  scrofulous  glands.  These 
glands  present  usually  a  slow  process,  are  commonly 
caseous,  and  always  possess  the  property  of  producing 
the  disease  when  introduced  into  the  tissues  of  suscepti- 
ble animals,  and  yet  they  are  the  most  difficult  of  all 
tissues  in  which  to  demonstrate  microscopically  the 
presence  of  tubercle  bacilli.  In  tubercles  containing 
giant-cells  the  bacilli  can  usually  be  demonstrated  in 
the  granular  contents  of  these  cells.  Frequently,  they 
will  be  found  accumulated  at  the  pole  of  the  cell  oppo- 
site to  that  occupied  by  the  nuclei,  as  if  there  existed 
an  antagonism  between  the  nuclei  and  the  bacilli.  In 
some  of  these  cells,  however,  the  distribution  of  the 
bacilli  is  seen  to  be  irregular  and  they  will  be  found 
scattered  among  the  nuclei  as  well  as  in  the  necrotic 
centre  of  the  cell. 

As  the  number  of  bacilli  in  the  giant-cell  increases 
the  cell  itself  is  ultimately  destroyed. 

Tubercular  tissues  always  contain  the  bacilli  or  their 
spores  and  are  always  capable  of  reproducing  the  dis- 
ease when  introduced  into  the  body  of  a  susceptible 
animal.  From  the  tissues  of  .this  animal  the  bacilli 
may  again  be  obtained  and  cultivated  artificially,  and 
these  cultures  are  capable  of  again  producing  the  dis- 
ease when  further  inoculated.  Thus  the  postulates 


CULTIVATION    OF    TUBERCLE    BACILLUS.    211 

which  are  necessary  to  prove  the  etiological  role  of  the 
organism  in  the  production  of  this  malady  are  all  ful- 
filled. 

THE  TUBERCLE  BACILLUS. — Of  the  three  pathogenic 
organisms  of  which  we  are  speaking,  the  tubercle  bacillus 
will  give  us  most  difficulty  in  our  efforts  at  cultivation. 

It  is  in  the  strict  sense  of  the  word  a  parasite  and 
finds  conditions  entirely  favorable  to  its  development 
only  in  the  animal  body.  On  ordinary  artificial 
media  the  bacilli  taken  directly  from  the  animal  body 
grow  only  very  imperfectly  or  in  many  cases  not  at  all. 
From  this  it  seems  probable  that  there  is  a  difference  in 
the  nature  of  the  individual  bacilli  of  this  group — some 
appearing  to  be  capable  only  of  growth  in  the  animal 
tissues,  while  others  are  apparently  possessed  of  the 
power  to  lead  a  limited  saprophytic  existence.  It  may  be, 
therefore,  that  those  bacilli  which  we  obtain  as  artificial 
cultures  from  the  animal  body  are  offsprings  from  the 
more  saprophytic  members  of  the  group.  At  best,  one 
never  sees  with  the  tubercle  bacillus  a  saprophytic  con- 
dition in  any  way  comparable  to  that  possessed  by  many 
of  the  other  organisms  with  which  we  have  to  deal. 

In  efforts  to  cultivate  this  organism  directly  from  the 
tissues  of  the  animal,  the  method  by  which  one  obtains  the 
best  results  is  that  recommended  by  Koch — cultivation 
upon  blood-serum.  So  strictly  is  this  organism  a  parasite 
that  very  limited  alterations  in  the  conditions  under  which 
it  is  growing  may  result  in  failure  to  successfully  study 
it.  It  is,  therefore,  necessary  that  the' injunctions  for 
obtaining  it  in  pure  culture  should  be  carefully  observed. 

The  blood-serum  upon  which  the  organism  is  to  be 
cultivated  should  be  comparatively  freshly  prepared — 
that  is,  it  should  not  be  dry. 


212  BACTERIOLOGY. 

PREPARATION  OF  CULTURES  FROM  TISSUES. — 
Under  strictest  antiseptic  precautions,  remove  from 
the  animal  the  tubercular  tissue — the  liver,  spleen,  or 
a  lymphatic  gland  being  preferable.  Place  the  tissue  in 
a  sterilized  Petri  dish  and  dissect  out  with  sterilized 
scissors  and  forceps  the  small  tubercular  nodules.  Place 
each  nodule  upon  the  surface  of  the  blood-serum,  one 
nodule  in  each  tube,  and  with  a  heavy,  sterilized,  looped 
platinum  needle  or  spatula,  rub  it  carefully  over  the  sur- 
face of  the  blood-serum.  It  is  best  to  dissect  away  twenty 
to  thirty  such  tubercles  and  treat  each  in  the  same  way. 
Some  of  the  tubes  will  remain  sterile,  others  may  be 
contaminated  by  outside  organisms  during  the  manipu- 
lation, while  a  few  may  give  the  result  desired — a 
growth  of  the  bacilli  themselves. 

After  inoculating  the  tubes  they  should  be  carefully 
sealed  up  to  prevent  evaporation  and  consequent  dry- 
ing. This  is  best  done  by  burning  oif  the  superfluous 
overhanging  cotton  plug  in  the  gas-flame,  and  then  im- 
pregnating the  upper  layers  of  the  cotton  with  either 
sealing-wax  or  paraffin  of  a  high  melting-point.  This 
precaution  is  necessary  because  of  the  slow  growth  of 
the  organism.  Under  the  most  favorable  conditions 
tubercle  bacilli  directly  from  the  animal  body  show 
no  evidence  of  growth  for  about  twelve  days  after 
inoculation  upon  blood-serum,  and,  as  they  must  be 
retained  during  this  time  at  the  body  temperature — 
37.5°  C. — evaporation  would  take  place  very  rapidly 
and  the  medium  become  too  dry  for  their  development. 

If  these  primary  efforts  result  in  the  appearance  of  a 
culture  of  the  bacilli,  further  cultivations  may  be  made 
by  taking  up  a  bit  of  the  colony,  preferably  a  moderately 
large  quantity,  and  transferring  it  to  fresh  serum,  and 


CULTURES    FROM    TISSUES.  213 

this  in  turn  is  sealed  up  and  retained  at  the  same  tem- 
perature. Once  having  obtained  the  organism  in  pure 
culture  its  subsequent  cultivation  may  be  conducted 
upon  the  glycerin-agar-agar  mixture — ordinary  neutral 
nutrient  agar-agar  to  which  6  or  7  per  cent,  of  glycerin 
has  been  added.  This  is  a  very  favorable  medium  for 
the  growth  of  this  organism  after  once  having  estab- 
lished its  saprophytic  form  of  existence,  though  blood- 
serum  is  perhaps  the  best  medium  to  be  employed  in 
obtaining  the  first  generation  of  the  organism  from  the 
tubercular  tissues. 

The  organism  may  be  cultivated  also  on  neutral  milk 
to  which  1  per  cent,  of  agar-agar  has  been  added,  also 
upon  the  surface  of  potato,  and  likewise  in  meat  infu- 
sion bouillon  to  which  6  or  7  per  cent,  of  glycerin  has 
been  added. 

In  appearance  the  cultures  of  the  tubercle  bacilli  are 
characteristic — after  once  having  seen  them  there  is  but 
little  probability  of  subsequent  mistake. 

They  appear  as  dry  masses,  which  may  develop  upon 
the  surface  of  the  medium  either  as  flat  scales  or  as 
clumps  of  mealy-looking  granules.  They  are  never 
moist,  and  frequently  have  the  appearance  of  coarse 
meal  which  had  been  spread  upon  the  surface  of  the 
medium.  In  the  lower  part  of  the  tube  in  which  they 
are  growing  (that  part  occupied  by  a  few  drops  of  fluid 
which  has  in  part  been  squeezed  from  the  medium 
during  the  process  of  solidification,  and  is  in  part  water 
of  condensation)  the  colonies  may  be  seen  to  float  as  a 
thin  pellicle  upon  the  surface. 

The  individuals  making  up  the  growth  adhere  so 
tenaciously  together  that  it  is  with  the  greatest  difficulty 
that  they  can  be  completely  separated.  In  even  the 

10* 


214:  BACTERIOLOGY. 

oldest  and  dryest  cultures  pulverization  is  impossible. 
The  masses  can  only  be  separated  and  broken  up  by 
grinding  in  a  mortar  with  the  addition  of  some  foreign 
substance,  such  as  very  fine,  sterilized  sand,  dust,  etc. 

The  cultures  are  of  a  dirty-drab  or  brownish-gray 
color  when  seen  on  serum  or  on  glycerin-agar-agar. 

On  potato  they  grow  in  practically  the  same  way, 
though  the  development  is  much  more  limited.  They 
are  here  of  nearly  the  same  color  as  the  potato  on  which 
they  are  growing. 

On  milk-agar-agar  they  are  of  so  nearly  the  same 
color  as  the  medium,  that  unless  they  are  growing  as  the 
mealy-looking  masses  considerably  elevated  above  the 
surface,  their  presence  is  less  conspicuous  than  when  on 
the  other  media. 

In  bouillon  they  appear  as  a  thin  pellicle  on  the  sur- 
face. This  may  fall  to  the  bottom  of  the  fluid  and  con- 
tinue to  develop,  its  place  on  the  surface  being  taken 
by  a  second  pellicle. 

Under  all  conditions  of  artificial  development  the 
cultures  of  this  organism  are  always  very  dry  and 
brittle  in  appearance,  though  in  truth  the  individuals 
adhere  tenaciously  together  by  a  very  glutinous  sub- 
stance. 

The  tubercle  bacillus  does  not  develop  on  gelatin, 
because  of  the  low  temperature  at  which  this  medium 
must  be  used. 

MICROSCOPIC  APPEARANCE  OF  THE  TUBERCLE 
BACILLUS. — Microscopically  the  organism  itself  is  a 
delicate  rod,  usually  somewhat  beaded  in  its  structure, 
though  rarely  it  is  seen  to  be  homogeneous.  It  is  either 
quite  straight  or  somewhat  curved  or  bent  on  its  long 
axis.  In  some  preparations  involution-forms,  consist- 


STAINING    PECULIARITIES.  215 

ing  of  rods  a  little  clubbed  at  one  extremity  or  slightly 
bulging  at  different  points,  may  be  detected.  It  varies 
in  length — sometimes  being  seen  in  very  short  seg- 
ments, again  much  longer.  On  an  average  its  length  is 
seen  to  vary  from  2  to  5  /w.  It  is  commonly  described 
as  being  in  length  about  one-fourth  to  one-half  the 
diameter  of  a  red  blood-corpuscle.  It  is  very  slender. 

These  rods  usually  present,  as  has  been  said,  an 
appearance  of  alternate  stained  and  colorless  portions. 
It  is  the  latter  portions  which  are  believed  to  be  the 
spores  of  the  organism,  though  as  yet  no  absolute  proof 
of  this  opinion  lias  been  established. 

At  times  these  colorless  portions  are  seen  to  bulge 
slightly  beyond  the  contour  of  the  rod,  and  in  this  way 
give  to  the  rods  the  beaded  appearance  so  commonly 
ascribed  to  them. 

STAINING  PECULIARITIES. — A  peculiarity  of  this 
organism  is  its  behavior  toward  staining  reagents,  and 
by  this  means  alone  it  may  be  easily  recognized.  The 
tubercle  bacilli  do  not  stain  by  the  ordinary  methods. 
They  possess  some  peculiarity  in  their  composition 
which  renders  them  more  or  less  proof  against  the 
simpler  dyes.  It  is  therefore  necessary  that  more  ener- 
getic and  penetrating  reagents  than  the  ordinary  watery 
solutions  should  be  employed  Experience  has  taught 
us  that  certain  substances  not  only  increase  the  solu- 
bility of  the  aniline  coloring  substances,  but  by  their 
presence  the  penetration  of  the  coloring  agents  is  very 
much  increased.  These  substances  are  aniline  oil  and 
carbolic  acid.  They  are  both  present  in  the  point  of 
the  solutions  to  about  saturation.  (For  the  exact 
proportions  see  chapter  on  Staining  Reagents.) 

Under  the  influence  of  heat,  these  solutions  are  seen 


216  BACTERIOLOGY. 

to  stain  all  bacteria  very  intensely — the  tubercle  bacilli 
as  well  as  the  ordinary  forms.  If  we  subject  our  prep- 
aration, which  may  contain  a  mixture  of  tubercle  bacilli 
and  other  forms,  to  the  action  of  decolorizing  agents, 
another  peculiarity  of  the  tubercle  bacilli  will  be  ob- 
served. While  all  other  organisms  in  the  preparation 
will  give  up  their  color  and  become  invisible,  the  tubercle 
bacilli  retain  it  with  marked  tenacity.  They  stain  with 
great  difficulty,  but  once  stained  they  retain  the  color 
even  under  the  influence  of  strong  decolorizing  agents. 

The  only  other  organism  possessing  a  similar  pecu- 
liarity is  the  bacillus  of  leprosy,  with  which,  under 
ordinary  conditions,  we  are  not  likely  to  come  in  con- 
tact. This  micro-chemical  reaction  therefore  serves  as 
a  means  of  differentiating  this  organism  in  sputum  and 
other  fluids  from  the  body  of  suspected  subjects  from 
all  other  bacteria  that  are  likely  to  be  present. 

SUSCEPTIBILITY  OF  ANIMALS  TO  TUBERCULOSIS. — 
The  animals  which  are  known  to  be  susceptible  to  the 
tubercular  processes  are  man,  apes,  cattle,  horses,  sheep, 
guinea-pigs,  pigeons,  rabbits,  cats,  and  field  mice. 

White  mice,  dogs,  and  rats  possess  immunity  against 
the  disease. 

We  have  reviewed  the  three  common  pathogenic 
organisms  with  which  we  may  come  in  contact  in  the 
sputum  of  tuberculous  individuals.  Occasionally  other 
forms  may  be  present.  The  pyogenic  forms  are  not 
rarely  found,  and  for  a  long  time  after  diphtheria  the 
bacillus  of  Lb'ffler  is  known  to  be  demonstrable  in  the 
pharynx.  These  latter  organisms  will  be  described 
under  their  proper  heads. 


CHAPTER    XXI. 

Suppuration — The  staphylococcus  pyogenes  aureus. 

PKEPAEE  from  the  pus  of  an  acute  abscess  or  boil, 
which  has  been  opened  under  antiseptic  precautions,  a 
set  of  plates  of  agar-agar.  Care  must  be  given  that 
none  of  the  antiseptic  fluid  gains  access  to  the  culture 
tubes,  otherwise  its  antiseptic  effect  may  be  seen  and  the 
development  of  the  organisms  interfered  with.  It  is 
best,  therefore,  to  take  up  a  drop  of  the  pus  upon  the 
platinum-wire  loop  after  it  has  been  flowing  for  a  few 
seconds ;  even  then  it  must  be  taken  from  the  mouth  of 
the  wound  and  before  it  has  run  over  the  surface  of  the 
skin.  At  the  same  time  prepare  two  or  three  cover-slips 
from  the  pus. 

Microscopic  examination  of  these  slips  will  reveal 
the  presence  of  a  large  number  of  pus-cells,  both  multi- 
nucleated  and  with  horseshoe-shaped  nuclei,  some  threads 
of  disintegrated  connective-tissue,  and,  lying  here  and 
there  throughout  the  preparation,  small  round  bodies 
which  will  sometimes  appear  singly,  sometimes  in  pairs, 
and  frequently  will  be  seen  grouped  together  somewhat 
after  the  manner  of  clusters  of  grapes.  They  stain 
readily  and  are  commonly  located  in  the  material  be- 
tween the  pus-cells  ;  very  rarely  they  may  be  seen  in  the 
protoplasmic  body  of  the  cell.  (Compare  the  preparation 
with  a  similar  one  made  from  the  pus  of  gonorrhoea.  In 
what  way  do  the  two  preparations  differ  the  one  from 
the  other?) 


218  BACTERIOLOGY. 

After  twenty-four  hours  in  the  incubator  the  plates 
will  be  seen  to  be  studded  here  and  there  with  yellow  or 
orange-colored  colonies,  which  are  usually  round,  moist, 
and  glistening  in  their  naked-eye  appearances.  Under 
the  low-power  hand-lens  they  are  frequently  irregularly 
star-shaped  or  lobulated  in  outline  and  appear  very  dense 
in  structure.  Under  the  low  objective  they  appear,  when 
on  the  surface,  as  coarsely  granular,  irregularly  round 
patches,  with  more  or  less  ragged  borders  and  a  dark 
irregular  central  mass,  which  has  somewhat  the  appear- 
ance of  masses  of  coarser  clumps  of  the  same  material 
as  that  composing  the  rest  of  the  colony.  When  deep 
down  in  the  culture  medium  they  present  but  little  that 
is  typical.  Microscopically,  these  colonies  are  composed 
of  small  round  cells,  irregularly  grouped  together.  They 
are  in  every  way  of  the  same  appearance  as  those  seen 
upon  the  cover- slip  preparations. 

Prepare  from  one  of  these  colonies  a  pure  stab  culture 
in  gelatin.  After  thirty-six  to  forty-eight  hours  lique- 
faction of  the  gelatin  along  the  track  of  the  needle,  and 
most  conspicuous  at  its  upper  end,  will  be  observed.  As 
growth  continues  the  liquefaction  becomes  more  or  less 
of  a  stocking-shape,  and  gradually  widens  out  at  its 
upper  end  into  an  irregular  funnel.  This  will  continue 
until  the  whole  of  the  gelatin  in  the  tube  eventually 
becomes  fluid.  There  can  always  be  noticed  at  the 
bottom  of  the  liquefying  portion  an  orange-colored  or 
yellow  mass  composed  of  a  number  of  the  organisms 
which  have  sunk  to  the  bottom  of  the  fluid. 

On  potato  the  growth  is  quite  luxuriant,  appearing  as 
a  brilliant  orange-colored  layer,  somewhat  lobulated  and 
a  little  less  moist  then  when  growing  upon  agar.  It 


SUPPURATION.  219 

does  not  produce  fermentation  with  gas-production.  It 
belongs  to  the  group  of  facultative  aerobes. 

In  milk  it  rapidly  brings  about  coagulation  with  acid 
reaction. 

It  is  not  motile,  and  being  of  the  family  of  micrococci, 
does  not  form  endogenous  spores.  It  possesses,  however, 
a  marked  resistance  toward  detrimental  agencies. 

In  bouillon  it  causes  a  diffuse  clouding,  and  after  a 
time  presents  a  yellow  sedimentation. 

This  organism  is  the  commonest  of  the  pathogenic 
bacteria  with  which  we  shall  meet.  It  is  the  staphy- 
lococcus  pyogenes  aureus,  and  is  the  organism  most 
frequently  concerned  in  the  production  of  acute,  cir- 
cumscribed, suppurative  inflammations.  It  is  almost 
everywhere  present,  and  is  the  organism  most  dreaded 
by  the  surgeon. 

In  studying  its  effects  upon  lower  animals  a  number 
of  points  are  to  be  remembered.  While  it  is  the  etio- 
logical  factor  in  the  production  of  most  of  the  suppura- 
tive processes  in  man,  still  it  is  with  no  little  difficulty 
that  these  conditions  can  be  reproduced  in  the  lower 
animals.  Its  subcutaneous  introduction  into  their  tis- 
sues does  not  always  result  in  abscess-formation,  and 
when  it  does  there  seems  to  have  been  some  coincident 
interference  with  the  circulation  in  these  tissues  which 
render  them  less  able  to  resist  its  inroads.  When 
introduced  into  the  great  serous  cavities  of  the  lower 
animals  it  is  not  always  followed  by  the  production 
of  inflammation.  If  the  abdominal  cavity  of  a  dog,  for 
example,  be  carefully  opened  so  as  to  make  as  slight  a 
wound  as  possible,  and  no  injury  be  done  to  the  intes- 
tines, large  quantities  of  bouillon  cultures  or  watery 
suspensions  of  this  organism  may,  and  repeatedly  have 


220  BACTERIOLOGY. 

been  introduced  into  the  peritoneum  without  the  slight- 
est injury  to  the  animal.  On  the  contrary,  if  some 
substance  which  acts  as  a  direct  irritant  to  the  intestines 
— such,  for  example,  as  a  small  bit  of  potato  upon  which 
the  organisms  are  growing — is  at  the  same  time  intro- 
duced, or  the  intestines  be  mechanically  injured,  so  that 
there  is  a  disturbance  in  their  circulation,  then  the  in- 
troduction of  these  organisms  is  promptly  followed  by 
acute  and  fatal  peritonitis. 

On  the  other  hand,  the  results  which  follow  their 
introduction  into  the  circulation  are  practically  constant. 
If  one  injects  into  the  circulation  of  the  rabbit  through 
one  of  the  veins  of  the  ear,  or  in  any  other  way,  from 
0.1  to  0.3  c.c.  of  a  bouillon  culture  or  watery  suspension 
of  this  organism,  a  fatal  pyaemia  always  follows  in  from 
two  and  one-half  to  three  days.  A  few  hours  before 
death  the  aniuial  is  frequently  seen  to  have  severe  con- 
vulsions. Now  and  then  excessive  secretion  of  urine  is 
noticed.  The  animal  may  appear  in  moderately  good 
condition  until  from  eight  to  ten  hours  before  death. 
At  the  autopsy  a  typical  picture  presents.  The  vol- 
untary muscles  are  seen  to  be  marked  here  and  there 
by  yellow  spots,  which  average  the  size  of  a  flax-seed, 
and  are  of  about  the  same  shape.  They  lie  usually  with 
their  long  axis  running  longitudinally  between  the  mus- 
cle fibres.  As  the  abdominal  aud  thoracic  cavities  are 
opened  the  diaphragm  is  not  rarely  seen  to  be  studded  by 
them.  Frequently  the  pericardial  sac  is  distended  with 
a  clear  gelatinous  fluid,  and  almost  constantly  the  yel- 
low points  are  to  be  seen  in  the  myocardium.  The  kid- 
neys are  rarely  without  them ;  here  they  appear  on  the 
surface  scattered  about  as  single  yellow  points,  or  again, 
are  seen  as  conglomerate  masses  of  small  yellow  points 


STUDY    OF    COVER-SLIPS    AND    SECTIONS.       221 

which  occupy,  as  a  rule,  the  area  fed  by  a  single  vessel. 
If  one  makes  a  section  into  one  of  these  yellow  points  it 
will  be  seen  to  extend  deep  down  through  the  substance 
of  the  kidney  as  a  yellow,  wedge-shaped  mass,  the  base 
of  the  wedge  being  at  the  surface  of  the  organ. 

It  is  very  rare  that  these  abscesses — for  abscesses  these 
yellow  points  are,  as  we  shall  see  when  we  come  to  study 
them  more  closely — are  found  either  in  the  liver,  spleen, 
or  brain ;  their  usual  location  being,  as  said,  in  the 
kidney,  myocardium,  and  voluntary  muscles. 

These  minute  abscesses  contain  a  dry,  cheesy,  necrotic 
centre,  in  which  the  staphylococci  are  present  in  large 
numbers,  as  may  be  seen  upon  cover-slips  prepared  from 
them.  They  may  also  be  obtained  in  pure  culture  from 
these  suppurating  points. 

Preserve  in  Miiller's  fluid  and  in  alcohol  duplicate 
bits  of  all  the  tissue  in  which  the  abscesses  are  located. 

When  these  tissues  are  hard  enough  to  cut,  sections 
should  be  made  through  the  abscess- points,  and  the 
histological  changes  carefully  studied. 

MICROSCOPIC  STUDY  OF  COVER-SLIPS  AND  SECTIONS. 
— In  cover  slip  preparations  this  organism  stains  readily 
with  the  ordinary  dyes. 

In  tissues,  however,  it  is  best  to  employ  some  method 
by  means  of  which  contrast  stains  may  be  employed, 
and  the  location  and  grouping  of  the  organisms  in  the 
tissues  rendered  more  conspicuous. 

When  stained,  sections  of  tissues  containing  these 
small  abscesses  present  the  following  appearances  : 

To  the  naked  eye  will  be  seen  here  and  there  in  the 
section,  if  the  abscesses  are  very  numerous,  small,  darkly 
stained  areas  which  range  in  size  from  that  of  a  pin- 
point up  to  those  having  a  diameter  of  from  I  to  2  mm. 


222  BACTERIOLOGY. 

These  points,  when  in  the  kidney,  may  be  round  or  oval 
in  outline,  or  may  appear  wedge-shaped,  with  the  base 
of  the  wedge  toward  the  surface  of  the  organ.  The 
differences  in  shape  depend  frequently  upon  the  direction 
in  which  the  section  has  been  made  through  the  kidney. 
In  the  muscles  they  are  irregularly  round  or  oval. 

When  quite  small  they  app?ar  to  the  naked  eye  as 
simple,  round  or  oval,  darkly  stained  points,  but  when 
they  are  more  advanced  a  pale  centre  can  usually  be 
made  out. 

When  magnified,  they  appear  in  the  earliest  stages 
as  minute  aggregations  of  small  cells,  the  nuclei  of 
which  stain  intensely.  Almost  always  there  can  be 
seen  about  the  centre  of  these  cell-accumulations  evi- 
dences of  progressing  necrosis.  The  normal  structure 
of  the  cells  of  the  tissue  will  be  more  or  less  destroyed ; 
there  will  be  seen  a  granular  condition  due  to  cell-frag- 
mentation; at  different  points  about  the  centre  of  this 
area  the  tissue  will  appear  cloudy  and  the  tissue-cells 
will  not  stain  readily.  All  about  and  through  this  spot 
will  be  seen  the  nuclei  of  pus  cells,  many  of  which  are 
undergoing  disintegration.  In  the  smallest  of  these 
beginning  abscesses  the  staphylococci  are  to  be  seen 
scattered  here  and  there  about  the  centre  of  the  necrotic 
tissue,  but  in  a  more  advanced  stage  they  are  commonly 
seen  massed  together  in  very  large  numbers  in  the  form 
commonly  referred  to  as  emboli  of  micrococci. 

The  localized  necrosis  of  the  tissues  which  is  seen  at 
the  centre  of  the  abscess  is  the  direct  result  of  the 
action  of  a  poison  produced  by  the  bacteria,  and  is  the 
starting-point  for  all  abscess- formations. 

When  the  process  is  somewhat  advanced  the  different 
parts  of  the  abscess  are  more  easily  detected.  They  then 
present  in  sections  somewhat  the  following  conditions : 


STUDY   OF    COVER-SLIPS    AND    SECTIONS.       223 

At  the  centre  can  be  seen  a  dense,  granular  mass,  which 
stains  readily  with  the  aniline  dyes  and,  wheu  highly 
magnified,  is  found  to  be  made  up  of  staphylococci.  Some- 
times the  shape  of  this  mass  of  staphylococci  corresponds 
to  that  of  the  capillary  in  which  the  organisms  became 
lodged  and  developed.  Immediately  about  the  embolus 
of  cocci  the  tissues  are  seen  to  be  in  an  advanced  stage 
of  necrosis.  Their  structure  is  almost  completely  de- 
stroyed, though  it  is  seen  to  be  more  advanced  in  some 
of  the  elements  of  the  tissues  than  in  others.  As  we 
approach  the  periphery  of  this  faintly  stained  necrotic 
area,  it  becomes  marked  here  and  there  with  granular 
bodies,  irregular  in  size  and  shape,  which  stain  in  the 
same  way  as  do  the  nuclei  of  the  pus-cells  and  represent 
the  result  of  disintregation  going  on  in  these  cells. 

Beyond  this  we  come  upon  a  dense,  deeply  stained 
zone,  consisting  of  closely  packed  pus-cells ;  of  granular 
detritus  resulting  from  destructive  processes  acting  upon 
these  cells ;  and  of  the  normal  cellular  and  connective- 
tissue  elements  of  the  part.  Here  and  there  through 
this  zone  will  be  seen  localized  areas  of  beginning  death 
of  the  tissues.  This  zone  gradually  fades  away  into  the 
healthy  surrounding  tissues.  It  constitutes  the  so-called 
"  abscess  wall." 

Such  is  the  picture  presented  by  the  miliary  abscess 
when  produced  experimentally  in  the  rabbit,  and  it 
corresponds  throughout  with  the  pathological  changes 
which  accompany  the  formation  of  larger  abscesses  in 
the  tissues  of  human  beings. 

From  these  small  abscesses  in  the  tissues  of  the  rabbit 
the  staphylococcus  pyogenes  aureus  may  again  be  ob- 
tained in  pure  culture,  and  will  present  identically  the 
same  characteristics  that  were  possessed  by  the  culture 
with  which  the  animal  was  inoculated. 


224  BACTERIOLOGY. 

THE  ,LESS  COMMON  PYOGENIC  ORGANISMS. — 
The  pus  of  an  acute  abscess  in  the  human  being  may 
sometimes  contain  other  organisms  beside  the  staphylo- 
coccus  pyogenes  aureus.  The  staphylococcus  pyogenes 
albus  and  citreus  may  be  found.  The  colonies  of  the 
former  are  white,  those  of  the  latter  are  lemon-color. 
The  streptococcus  pyogenes  is  also  sometimes  present. 
The  commonest  of  the  pyogenic  organisms  however  is 
that  just  described — the  staphylococcus  pyogenes  aureus. 

THE  STREPTOCOCCUS  PYOGENES. — From  a  spreading 
phlegmonous  inflammation  prepare  cultures.  What  is 
the  predominating  organism  ?  Does  it  appear  as  irregu- 
lar clusters  of  grapes,  or  has  its  individuals  a  definite 
regular  arrangement?  Are  its  colonies  like  those  of  the 
staphylococcus  pyogenes  aureus  ? 

Isolate  this  organism  in  pure  cultures.  In  these  cul- 
tures it  will  be  found  to  present  an  arrangement  some- 
what like  a  chain  of  beads.  Determine  its  cultural 
peculiarities  and  describe  them  accurately. 

This  is  the  streptococcus  pyogenes,  and  is  the  organ- 
ism most  commonly  found  in  rapidly  spreading  suppu- 
ration in  contradistinction  to  the  staphylococcus  pyogenes 
aureus,  which  is  most  frequently  found  in  circumscribed 
abscess- format  ions  ;  they  may  be  found  together. 

If  the  opportunity  presents,  obtain  cultures  from  a 
case  of  erysipelas.  Compare  the  organism  thus  obtained 
with  the  streptococcus  just  mentioned.  Inoculate  a  rabbit 
both  subcutaneously  and  into  the  circulation  with  about 
0.2  c.c.  of  a  pure  bouillon  culture  of  these  organisms. 
Do  the  results  correspond,  and  do  they  in  any  way 
suggest  the  results  obtained  with  the  staphylococcus 
pyogenes  aureus  when  introduced  into  animals  in  the 
same  way?  Do  these  streptococci  flourish  readily  on 
ordinary  media? 


CHAPTER    XXII. 

Typhoid  fever — Study  of  the  organism  concerned  in  its  production. 

THE  organism,  discovered  by  Eberth  and  by  Gaffky, 
which  is  recognized  as  the  etiological  factor  in  the 
production  of  typhoid  fever,  may  be  described  as 
follows  : 

In  patients  suffering  from  this  disease  it  has  been 
found  during  life  in  the  blood,  urine,  and  feces,  and  at 
autopies  in  the  tissues  of  the  spleen,  liver,  kidneys, 
intestinal  lymphatic  glands,  and  intestines. 

It  is  a  bacillus  about  three  times  as  long  as  it  is  broad, 
with  rounded  ends.  It  may  appear  at  one  time  as  very 
short  ovals,  at  another  time  as  long  threads.  Its  breadth 
remains  tolerably  constant.  Its  morphology  presents 
nothing  that  will  aid  in  its  identification.  It  stains  a 
little  less  readily  with  the  aniline  dyes  than  do  most  of 
the  other  organisms.  It  is  very  actively  motile,  and 
when  stained  by  the  special  method  of  Loffler  (see  this 
method  in  chapter  on  Stainings)  is  seen  to  possess  very 
delicate  locomotive  organs  in  the  form  of  fine,  hair-like 
flagellse,  which  are  given  off  in  large  numbers  from  all 
parts  of  its  surface.  These  flagellse  are  not  seen  in 
unstained  preparations,  or  are  they  rendered  visible  by 
the  ordinary  methods  of  staining, 

GELATIN  PLATES. — Its  growth,  when  seen  in  the 
depths  of  the  medium,  has  nothing  characteristic,  ap- 
pearing simply  as  round  or  oval,  finely  granular  points. 
On  the  surface  it  develops  as  very  superficial,  blue-white 


226  BACTERIOLOGY. 

colonies,  with  irregular  borders.  They  are  a  little  denser 
at  the  centre  than  at  the  periphery.  When  magnified, 
the  colonies  present  wrinkles  or  folds,  which  give  to 
them,  in  miniature,  the  appearanoe  seen  in  the  relief 
maps  which  represent  mountainous  districts.  These 
colonies  have  sometimes  the  appearance  of  flattened 
pellicles  of  glass-wool,  and  glisten  with  more  or  less 
of  a  bronze  color. 

ON  AGAR-AGAR  the  colonies  present  nothing  typical. 

STAB  CULTURES. — In  stab  cultures  the  growth  is 
mostly  on  the  surface,  there  being  only  a  very  limited 
development  down  the  track  made  by  the  needle.  The 
surface  growth  has  the  same  appearance  in  general  as 
that  given  for  the  colonies. 

POTATO. — The  growth  on  potato  is  usually  described 
as  luxuriant  but  invisible,  making  its  presence  evident 
only  by  the  production  of  a  slight  increase  of  moisture 
at  the  inoculated  point,  and  by  a  limited  resistance 
offered  to  the  needle  when  scraped  across  the  track  of 
growth. 

POTATO  GELATIN. — The  growth  is  similar  to  that 
upon  ordinary  nutrient  gelatin. 

MILK. — It  does  not  cause  coagulation  when  grown  in 
sterilized  milk. 

It  does  not  liquefy  gelatin. 

It  grows  both  with  and  without  oxygen. 

It  does  not  produce  indol. 

In  bouillon  it  causes  a  uniform  clouding  of  the 
medium  and  brings  about  a  slightly  acid  reaction. 

It  does  not  grow  rapidly. 

It  does  not  produce  fermentation  with  liberation  of  gas. 

It  does  not  form  spores.  The  irregularities  of  stain- 
ing so  commonly  seen  in  this  organism  have  in  some 


TYPHOID    BACILLI    IN    TISSUES.  227 

instances  led  to  the  belief  that  the  pale,  unstained  portions 
of  the  bacilli  indicate  the  presence  of  spores.  More 
exact  tests,  however,  have  demonstrated  the  error  of  this 
opinion. 

It  grows  at  any  temperature  between  20°  C.  and 
38°  C.,  though  more  favorably  at  the  latter  point. 

It  is  very  sensitive  to  high  temperatures,  being  killed 
by  an  exposure  of  ten  minutes  to  60°  C.,  and  in  a  much 
shorter  time  to  slightly  higher  temperatures. 

Owing  to  a  tendency  toward  retraction  of  its  proto- 
plasm from  the  cell  envelope  and  the  consequent  pro- 
duction of  vacuoles  in  the  bacilli,  the  staining  of  this 
organism  is  usually  more  or  less  irregular.  At  some 
points  in  a  single  cell  marked  differences  in  the  intensity 
of  the  staining  will  be  seen,  and  here  and  there  areas 
quite  free  from  color  can  commonly  be  detected. 

PRESENCE  IN  TISSUES. — It  is  not  easy  to  demonstrate 
this  organism  in  tissues  unless  it  is  present  in  large  num- 
bers. The  manipulations  to  which  the  sections  are  sub- 
jected in  being  mounted  rob  the  bacilli  of  their  staining, 
and  render  them  invisible,  or  nearly  so.  If,  however, 
sections  be  stained  in  the  carbolic- fuch sin  solution,  either 
at  the  ordinary  temparature  of  the  room  for  from  eigh- 
teen to  twenty  hours,  or  at  a  higher  temperature 
(40°  to  45°  C.)  for  a  shorter  time,  then  washed  out  in 
absolute  alcohol,  and  cleared  up  in  xylol  and  mounted 
in  balsam,  as  a  rule,  the  bacilli  (particularly  if  the  tissue 
is  the  liver  or  spleen)  can  readily  be  detected  massed 
together  in  their  characteristic  clumps.  If  used  in  the 
same  way,  the  methylene-blue  solution  gives  also  very 
satisfactory  results. 

In  searching  for  the  typhoid  bacilli  in  tissues,  their 


228  BACTERIOLOGY. 

mode  of  growth  under  these  circumstances  must  always 
be  borne  in  mind,  otherwise  much  labor  will  be  ex- 
pended in  vain.  In  tissues  the  typhoid  bacilli  do  not 
lie  scattered  about  in  the  same  way  as  do  the  organisms 
in  tissues  from  cases  of  septicaemia.  They  are  not 
regularly  distributed  along  the  course  of  the  capillaries  ; 
they  are  localized  in  small  clumps  through  the  tissues, 
and  it  is  for  thesa  clumps,  which  are  easily  detected 
under  the  low-power  objective,  that  one  should  search. 
When  the  section  is  prepared  for  examination,  if  it  is 
gone  over  with  the  low-power  objective,  one  will  notice 
here  and  there  little  masses  that  look  in  every  respect 
like  particles  of  staining-matter  which  have  been  pre- 
cipitated upon  the  section  at  that  point.  If  these  little 
masses  are  examined  with  a  higher  power  objective, 
they  will  be  found  to  consist  of  small  ovals  or  short 
rods  so  closely  packed  together  that  the  individuals 
composing  the  clump  can  be  seen  only  at  the  very 
periphery  of  the  mass.  This  is  the  characteristic  ap- 
pearance of  the  typhoid  organism  in  tissues.  The 
little  masses  are  usually  in  the  neighborhood  of  a 
capillary. 

RESULT  OF  INOCULATION  INTO  LOWER  ANIMALS. — 
A  great  many  experiments  have  been  made  with  the 
view  of  reproducing  the  pathological  conditions  of  this 
disease,  as  seen  in  man,  in  the  tissues  of  lower  animals, 
but  with  limited  success.  Fatal  results  without  the 
appearance  of  the  typical  pathological  changes  have  fre- 
quently followed  these  attempts,  but  in  most  cases  they 
could  be  easily  traced  to  the  toxic,1  rather  than  to  the 

1  Toxic — poisonous  results  not  necessarily  accompanied  by  the 
growth  of  organisms  in  the  tissues. 


TYPHOID    INOCULATION    IN    ANIMALS.      229 

truly  infective1  action  of  the  materials  introduced  into 
the  animals. 

The  most  successful  efforts  for  the  production  of 
the  typical  typhoid  lesions  in  lower  animals  are  those 
recently  reported  by  Cygnasus.  By  the  introduction 
of  the  typhoid  bacilli  into  the  tissues  of  dogs,  rabbits, 
and  mice  he  was  able  to  produce  in  the  small  intestines 
conditions  which  were  histologically  and  to  the  naked 
eye  analogous  to  those  found  in  the  human  subject. 

Of  a  number  of  experiments  made  by  the  writer 
with  the  same  object  in  view,  ouly  one  positive  result 
followed  the  introductiou  of  typhoid  bacilli  into  the 
circulation  of  rabbits.  In  this  case  the  ulcer  in  the 
ileum  was  macroscopically  and  microscopically  identical 
with  those  found  at  autopsy  in  the  small  intestine  of 
the  human  subject  dead  of  this  disease.  The  typhoid 
bacilli  were  not  only  obtained  from  the  spleen  of  the 
animal  by  culture  methods,  but  were  also  demonstrated 
microscopically  in  their  characteristic  clumps  in  sections 
of  the  organ. 

Because  of  the  variations  iu  the  morphology  and  cul- 
tural peculiarities  of  this  organism,  and  because  of  the 
difficulty  experienced  in  efforts  to  reproduce  in  lower 
animals  the  conditions  found  in  the  human  subject, 
typhoid  fever  is  bacteriologically  one  of  the  most  un- 
satisfactory of  the  infectious  diseases. 

There  are  a  number  of  other  organisms  which  botani- 
cally  appear  to  be  so  closely  related  to  the  typhoid  bacil- 
lus, and  which,  with  our  present  methods  for  studying 
these  organisms,  so  closely  simulate  it,  that  the  difficulty 

1  Infective  or  septic — poisoning  of  the  tissues  as  a  result  of  the 
growth  of  bacteria  in  them. 

11 


230  BACTERIOLOGY. 

of  identifying  this  organism  is  very  great.  In  addition  to 
this,  the  variability  constantly  seen  in  pure  cultures  of 
the  typhoid  bacillus  itself  in  no  way  renders  the  task 
more  simple. 

For  example,  the  morphology  of  the  typhoid  bacillus 
is  conspicuously  inconstant;  its  growth  on  potato,  which 
is  usually  given  as  characteristic,  may,  with  the  same 
culture,  at  one  time  appear  as  the  typical  invisible  de- 
velopment, at  another  time  it  may  grow  in  a  way  easily 
to  be  seen  with  the  naked  eye ;  the  change  of  reaction 
which  it  is  said  to  produce  in  bouillon  is  sometimes 
much  more  intense  than  at  others. 

The  only  properties  possessed  by  it  that  may  be  said 
to  be  constant  are  its  motility,  the  absence  of  indol- 
production,  and  its  growth  on  gelatin  plates ;  but  there 
are  other  organisms  which  possess  these  same  character- 
istics in  a  degree  that  renders  their  differentiation  from 
the  typhoid  organism  a  matter  of  extreme  difficulty,  if 
not  of  impossibility. 

These  points  should  be  borne  in  mind  in  the  exami- 
nation of  drinking-water  supposed  to  be  contaminated 
by  typhoid  dejections,  for  the  organisms  which  most 
nearly  approach  the  typhoid  bacillus  in  growth  and 
morphology  are  just  those  organisms  which  would 
appear  in  water  contaminated  from  cesspools,  i.  e.,  the 
organisms  constantly  found  in  the  normal  intestinal 
tract.  Even  in  the  stools  of  typhoid- fever  patients 
the  presence  of  these  normal  inhabitants  of  the  intes- 
tinal tract  renders  the  isolation  of  the  typhoid  organisms 
no  small  task. 

The  spleen  of  a  patient  dead  of  typhoid  fever  is  the 
safest  place  from  which  to  obtain  cultures  of  this  organ- 
ism for  study.  But  it  must  always  be  remembered  that 


TYPHOID    INOCULATION    IN    ANIMALS.      231 

even  here  the  bacterium  coli  communis,  the  normal 
organism  of  the  colon,  is  not  unlikely  to  be  found.  This 
organism,  however,  always  grows  visibly  on  potato,  co- 
agulates milk,  produces  a  more  pronounced  pink  color  in 
litmus-milk  than  does  the  typhoid  bacillus,  and  is  much 
less  actively  motile. 

Obtain  a  pure  culture  of  typhoid  bacilli  and  from 
this  make  inoculations  upon  a  series  of  potatoes  of  dif- 
ferent age  and  from  different  sources.  Do  they  all  grow 
alike  ? 

Make  a  series  of  twelve  tubes  of  peptone  solution  to 
which  rosolic  acid  has  been  added.  Inoculate  them  all 
with  as  near  the  same  amount  of  material  as  possible 
(one  loopful  from  a  bouillon  culture  into  each  tube) ; 
place  them  all  in  the  incubator.  Is  the  color-change,  as 
compared  with  the  control  tube,  the  same  in  all  cases? 

Compare  the  morphology  of  cultures  of  the  same  age 
on  gelatin,  agar-agar,  and  potato. 

Select  a  culture  in  which  the  vacuolations  are  quite 
marked.  Examine  this  culture  unstained.  Do  the  or- 
ganisms look  as  if  they  contained  spores  ?  How  would 
you  demonstrate  that  the  vacuolations  are  not  spores  ? 

Obtain  from  the  normal  feces  a  pure  culture  of  the 
commonest  organism  present.  Write  a  full  description 
of  it.  Now  make  parallel  cultures  of  this  organism  and 
of  the  typhoid  organism  on  all  the  different  media.  How 
do  they  differ  ?  In  what  respects  are  they  similar  ? 


CHAPTER    XXIII. 

Study  of  the  bacillus  of  anthrax,  and  the  effects  produced  by  its 
inoculation  into  animals  —  Peculiarities  of  the  organism  under 
varying  conditions  of  surroundings. 

THE  discovery  that  the  blood  of  animals  suffering 
from  splenic  fever,  or  anthrax,  always  contained  minute 
rod  shaped  bodies  (Pollender,  1855;  Davaine,  1863), 
led  to  a  closer  study  of  this  disease,  and  has  resulted, 
probably,  in  contributing  more  to  our  knowledge  of 
bacteriology  in  general  than  work  upon  any  of  the 
other  infectious  maladies. 

The  outcome  of  these  investigations  is  that  a  rod- 
shaped  microorganism,  now  known  as  the  bacillus  of 
anthrax,  is  always  present  in  the  blood  of  animals  suf- 
fering from  this  disease;  that  this  organism  can  be  ob- 
tained from  the  tissues  of  these  animals  in  pure  cultures, 
and  that  these  artificial  cultures  of  the  bacillus  of  anthrax 
when  introduced  into  the  body  of  susceptible  animals 
can  again  produce  a  condition  identical  to  that  found  in 
the  animal  from  which  they  were  obtained. 

The  disease  is  a  true  septicaemia,  and  the  capillaries 
throughout  the  body  after  death  will  always  be  found 
to  contain  the  typical  rod-shaped  organism  in  larger  or 
smaller  numbers. 

This  organism,  when  isolated  in  pure  cultures,  is  seen 
to  be  a  bacillus  which  varies  considerably  in  its  length, 
ranging  from  short  rods  of  2  to  3  ^  in  length  to  longer 
rods  of  20  to  25  /*  in  length.  In  breadth  it  is  from  1 


STUDY    OF    THE    BACILLUS    OF    ANTHRAX.      233 

to  1.25  p.  Frequently  very  long  threads  made  up  of 
several  rods,  joined  end  to  end,  are  seen. 

As  it  is  obtained  from  the  body  of  the  animal,  it  is 
usually  in  the  form  of  short  rods  square  at  the  ends. 

When  cultivated  artificially  at  the  temperature  of  the 
body,  the  bacillus  of  anthrax  presents  a  series  of  very 
interesting  stages. 

The  short  rods  develop  into  long  threads,  which  may 
be  seen  twisted  or  plaited  together  after  the  manner  of 
ropes,  each  thread  being  marked  by  the  points  of  junc- 
tion of  the  short  rods  composing  it. 

In  this  condition  it  remains  until  alterations  in  its 
surroundings,  most  conspicuously  diminution  in  its  nutri- 
tive supply,  favor  the  production  of  spores.  When  this 
stage  begins,  alterations  in  the  protoplasm  of  the  bacilli 
may  be  noticed;  they  become  marked  by  irregular,  gran- 
ular bodies,  which  eventually  coalesce  into  glistening, 
oval  spores,  one  of  which  lies  in  nearly  every  segment 
of  the  long  thread,  and  gives  to  the  thread  the  appear- 
ance of  a  string  of  glistening  beads.  In  this  stage  they 
remain  but  a  short  time.  The  chains  of  spores,  which 
are  held  together  by  the  remains  of  the  cells  in  which 
they  formed,  become  broken  up,  and  eventually  nothing 
but  free  oval  spores,  and  here  and  there  the  remains  of 
mature  bacilli  which  have  undergone  degenerative 
changes,  can  be  found.  In  this  condition  the  spores, 
capable  of  resisting  deleterious  influences,  remain  and, 
unless  their  surroundings  are  altered,  have  been  seen  to 
continue  in  this  living,  though  inactive,  condition  for  a 
very  long  time.  When  placed  under  favorable  condi- 
tions again,  each  spore  will  germinate  into  a  mature  cell, 
and  the  same  series  of  changes  will  be  repeated  until 
the  favorable  surroundings  become  again  gradually 


234  BACTERIOLOGY. 

unfavorable  to  development,  when  the  spore-formation 
is  again  seen.  The  spore-formation  takes  place  only  at 
temperatures  ranging  from  18°  to  43°  C.,  37.50°  C. 
being  the  most  favorable  temperature.  Under  12°  C. 
they  are  not  formed.  With  this  organism,  spore- forma- 
tion does  not  occur  in  the  tissues  of  the  living  animal, 
its  usual  condition  at  this  time  being  that  of  short  rods. 
Occasionally,  however,  somewhat  longer  forms  may  be 
seen. 

The  bacillus  of  anthrax  is  not  motile. 

GROWTH  ON  AGAR-AGAR. — The  colonies  of  this  or- 
ganism, as  seen  upon  agar-agar,  present  a  very  typical 
appearance,  from  which  they  have  been  likened  unto 
the  hair  of  a  Medusa.  From  a  central  point  which  is 
more  or  less  dense,  consisting  of  a  felt-like  mass  of  long 
threads  matted  irregularly  together,  the  growth  continues 
outward  upon  the  surface  of  the  agar.  It  is  made  up  of 
wavy  bundles  in  which  the  threads  are  seen  to  lie  parallel 
side  by  side  or  are  twisted  in  strands  like  those  of  a  rope 
— sometimes  they  have  a  plaited  arrangement.  These 
bundles  twist  about  and  cross  in  all  directions,  and 
eventually  disappear  at  the  periphery  of  the  colony. 
The  colony  itself  is  not  circumscribed  in  its  appearance, 
but  is  more  or  less  irregularly  fringed  and  ragged,  or 
scalloped.  To  the  naked  eye  they  look  very  much  like 
minute  pellicles  of  raw  cotton  which  had  been  pressed 
into  the  surface  of  the  agar-agar.  At  the  extreme  peri- 
phery of  the  colonies  it  is  sometimes  possible  to  trace 
single  bundles  of  these  threads  for  long  distances  across 
the  surface  of  the  agar-agar. 

As  the  colonies  continue  to  grow,  they  become  more 
and  more  dense ;  they  become  opaque  in  color,  and  gran- 
ular and  rough  on  the  surface.  When  touched  with  a 


ANTHRAX    STAB    AND    SLANT    CULTURES.    235 

sterilized  needle,  one  experiences  a  sensation  that  sug- 
gests, somewhat,  the  matted  structure  of  these  colonies. 
The  bit  that  may  thus  be  taken  from  a  colony  is  always 
more  or  less  ragged. 

GELATIN. — The  colonies  on  gelatin  at  the  earliest 
stages  also  present  the  same  wavy  appearance ;  but 
this  characteristic  soon  becomes  in  part  destroyed  by 
the  liquefaction  of  the  gelatin  which  is  produced  by 
the  growing  organisms.  This  allows  them  to  sink  to 
the  bottom  of  the  fluid,  where  they  lie  as  an  irregular 
mass.  Through  the  fluid  portion  of  the  gelatin  may  be 
seen  small  clumps  of  growing  bacilli,  which  look  very 
much  like  bits  of  cotton-wool . 

BOUILLON. — In  bouillon  the  growth  is  characterized 
by  the  formation  of  flaky  masses,  which  also  have  very 
much  the  appearance  of  bits  of  cotton.  Microscopic 
examination  of  one  of  these  flakes  shows  the  twisted 
and  plaited  arrangement  of  the  long  threads. 

POTATO. — It  develops  rapidly  as  a  dry,  granular, 
whitish  mass,  which  is  more  or  less  limited  to  the  point 
of  inoculation.  On  potato,  at  the  temperature  of  the 
incubator,  its  spore- formation  may  easily  be  observed. 

STAB  AND  SLANT  CULTURES. — Stab  and  slant  cul- 
tures on  agar-agar  present  in  general  the  appearances 
given  for  the  colonies,  except  that  the  growth  is  much 
mqre  extensive.  The  growth  is  always  more  pronounced 
on  the  surface  than  down  the  track  of  the  needle. 

On  gelatin  it  causes  liquefaction,  which  begins  on  the 
surface  at  the  point  inoculated,  and  spreads  outward  and 
downward. 

It  grows  best  with  access  to  oxygen,  and  very  poorly 
when  the  supply  of  oxygen  is  interfered  with. 


236  BACTERIOLOGY. 

Under  favorable  conditions  of  oxygen,  nutrition,  and 
temperature  its  growth  is  rapid. 

Uuder  12°  C.  and  above  45°  C.  no  growth  occurs. 
The  temperature  of  the  body  is  most  favorable  to  its 
development.  The  spores  of  the  anthrax  bacillus  are 
very  resistant  to  heat,  though  the  degree  of  resistance  is 
seen  to  vary  with  spores  of  different  origin.  Esmarch 
found  that  anthrax  spores  from  one  source  would  read- 
ily be  killed  by  an  exposure  of  one  minute  to  the  tem- 
perature of  steam,  whereas  those  from  other  sources 
resisted  this  temperature  for  longer  times,  reaching  in 
some  cases  as  long  as  twelve  minutes. 

STAINING. — The  anthrax  bacilli  stain  readily  with 
the  ordinary  aniline  dyes.  In  tissues  their  presence 
may  also  be  demonstrated  by  the  ordinary  aniline  stain- 
ing fluids,  or  by  Gram's  method.  They  may  also  be 
stained  in  tissues  with  a  strong  watery  solution  of  dahlia, 
after  which  the  tissue  is  decolorized  in  2  per  cent,  soda 
solution,  washed  in  water,  dehydrated  in  alcohol,  cleared 
up  in  xylol,  and  mounted  in  balsam.  This  leaves  the 
bacilli  stained,  while  the  tissues  are  decolorized ;  or  the 
tissues  may  be  stained  a  contrast  color — eosin,  for  ex- 
ample— after  the  dehydration  in  alcohol,  and  before  the 
clearing  up  in  xylol.  In  this  case  they  must  be  washed 
out  again  in  alcohol  before  using  the  xylol.  In  the 
preparation  treated  in  this  way,  the  rod-shaped  organ- 
isms will  be  of  a  purple  color,  and  will  be  seen  in  the 
capillaries  of  the  tissues,  while  the  tissues  themselves 
will  be  of  a  pale-rose  color. 

INOCULATION  INTO  ANIMALS. — Introduce  into  the 
subcutaneous  tissues  of  the  abdominal  wall  of  a  guinea- 
pig  or  rabbit,  a  portion  of  a  pure  culture  of  the  bacillus 
anthracis.  In  about  forty- eight  hours  the  animal  will 


ANTHKAX    INOCULATION    IN    ANIMALS.    237 

be  found  dead.  Immediately  at  the  point  of  inoculation 
but  little  or  no  reaction  will  be  noticed,  but  beyond 
this,  extending  for  a  long  distance  over  the  abdomen 
and  thorax,  the  tissues  will  be  markedly  oadematous. 
Here  and  there,  scattered  through  this  oedematous  tissue, 
small  ecchymoses  will  be  seen.  The  underlying  muscles 
are  pale  in  color.  Inspection  of  the  internal  viscera 
reveals  no  very  marked  macroscopic  changes  except  in 
the  spleen.  This  is  enlarged,  dark  in  color,  soft  and 
brittle.  The  liver  may  present  the  appearance  of  cloudy 
swelling ;  the  lungs  may  be  pale  or  pale-red  in  color ;  the 
heart  is  usually  filled  with  blood.  There  are  no  other 
changes  to  be  seen  by  the  naked  eye.  Prepare  cover- 
slip  preparations  from  the  blood  and  other  viscera. 
They  will  all  be  found  to  contain  the  short  rods  in 
large  numbers.  Nowhere  can  spore-formation  be  de- 
tected. Upon  microscopic  examination  of  sections  of  the 
organs  which  have  been  hardened  in  alcohol,  the  capilla- 
ries are  seen  to  be  filled  with  the  bacilli ;  in  some  places 
closely  packed  together  in  large  numbers,  at  other  points 
fewer  in  number.  Usually  they  are  present  in  largest 
numbers  in  those  tissues  having  the  greatest  capillary 
distribution  and  at  those  points  at  which  the  circulation  is 
slowest.  They  are  moderately  evenly  distributed  through 
the  spleen.  The  glomeruli  of  the  kidneys  and  the  capilla- 
ries of  the  lungs  are  frequently  quite  packed  with  them. 
The  capillaries  of  the  liver  contain  them  in  large  numbers. 
Hemorrhages,  probably  due  to  rupture  of  capillaries  by 
the  mechanical  pressure  of  the  bacilli  which  are  develop- 
ing in  them,  not  uncommonly  occur.  When  this  occurs 
in  the  mucous  membranes  of  the  alimentary  tract,  the 
blood  may  escape  through  the  mouth  or  anus  ;  when  in 
the  kidneys,  through  the  uriniferous  tubules. 

11* 


238  BACTERIOLOGY. 

Cultures  from  the  different  organs  or  from  the  oedema- 
tons  fluid  about  the  point  of  inoculation,  result  in  growth 
of  the  bacillus  anthracis. 

The  amphibia,  dogs,  and  the  majority  of  birds  are 
not  susceptible  to  this  disease.  Eats  are  difficult  to  infect. 
Rabbits,  guinea-pigs,  white  mice,  gray  house-mice,  sheep, 
and  cattle  are  susceptible.  Infection  may  occur  either 
through  the  circulation,  through  the  air-passages,  through 
the  alimentary  tract,  or,  as  we  have  just  seen,  through 
the  subcutaneous  tissues. 

EXPERIMENTS. 

Prepare  three  cultures  of  anthrax  bacilli — one  upon 
gelatin,  one  upon  agar-agar,  and  one  upon  potato.  Allow 
the  gelatin  culture  to  remain  at  the  ordinary  temperature 
of  the  room,  place  the  agar  culture  in  the  incubator,  and 
the  potato  culture  at  a  temperature  not  above  18°  to 
20°  C.  Prepare  cover-slips  from  each  from  day  to  day. 
What  differences  are  observed  ? 

Prepare  two  potato  cultures  of  the  anthrax  bacillus. 
Place  one  in  the  incubator  and  retain  the  other  at  a  tem- 
perature of  from  18°  to  20°  C.  Examine  them  each 
day.  Do  they  develop  in  the  same  way  ? 

From  a  fresh  culture  of  anthrax  bacilli,  in  which 
spore-formation  is  not  yet  begun,  prepare  a  hanging- 
drop  preparation ;  also  a  cover-slip  preparation  in  the 
usual  way  and  stain  it  with  a  strong  gentian- violet  solu- 
tion, and  another  cover-slip  preparation  which  will  be 
drawn  through  the  flame  twelve  to  fifteen  times,  stained 
with  aniline  gentian-violet,  washed  off  in  iodine  solu- 


EXPERIMENTS.  239 

tion,  and  then  in  water.  Examine  these  microscopically. 
Do  they  all  present  the  same  appearance  ?  To  what  are 
the  differences  due  ? 

Do  the  anthrax  threads,  as  seen  in  a  fresh,  growing 
hanging  drop,  present  the  same  morphological  appear- 
ance as  when  dried  and  stained  upon  a  cover-slip? 
How  do  they  differ? 

Liquefy  a  tube  of  agar-agar,  and  when  it  is  at  the 
temperature  of  40°  to  43°  C.,  add  a  very  minute  quan- 
tity of  an  anthrax  culture  which  is  far  advanced  in  the 
spore  stage.  Mix  it  thoroughly  with  the  liquid  agar- 
agar  and  from  this  prepare  several  hanging  drops  under 
strict  antiseptic  precautions,  using  the  fluid  agar-agar 
for  the  drops  instead  of  bouillon  or  salt  solution.  Select 
from  among  these  preparations  that  one  in  which  the 
smallest  number  of  spores  are  present.  Under  the 
microscope  observe  the  development  of  this  spore  into  a 
mature  cell.  Describe  carefully  the  steps. 

Prepare  a  1  : 1000  solution  of  carbolic  acid  in  bouillon. 
Inoculate  this  with  virulent  anthrax  spores.  If  no  de- 
velopment occurs  after  two  or  three  days  at  the  tempera- 
ture of  the  thermostat,  prepare  a  solution  of  1  : 1200, 
and  continue  until  the  point  is  reached  at  which  the 
amount  of  carbolic  acid  present  just  admits  of  the  de- 
velopment of  the  spores.  When  the  proper  dilution  is 
reached  prepare  a  dozen  of  such  tubes  and  inoculate  one 
of  them  with  virulent  anthrax  spores.  As  soon  as  de- 
velopment is  well  advanced  transfer  a  loopful  from  this 
tube  into  a  second  of  the  carbolic  acid  tubes ;  when  this 
has  developed,] then  from  this  into  a  third,  etc.  After 


240  BACTERIOLOGY. 

five  or  six  generations  which  have  been  treated  in  this 
way,  study  the  spore-production  of  the  organisms  in 
that  tube.  If  it  is  normal,  continue  to  inoculate  from 
one  carbolic  acid  tube  into  another,  and  see  if  it  is 
possible  by  this  means  to  influence  in  any  way  the 
production  of  spores  by  the  organism  with  which  you 
are  working.  What  is  the  effect,  if  any  ? 

Prepare  two  bouillon  cultures,  each  from  one  drop  of 
blood  of  an  animal  dead  of  anthrax.  Allow  one  of  them 
to  grow  for  from  fourteen  to  eighteen  hours  in  the  incu- 
bator ;  the  other  allow  to  grow  at  the  same  temperature 
for  three  or  four  days.  Remove  the  first  after  the  time 
mentioned  and  subject  it  to  a  temperature  of  80°  C.  for 
thirty  minutes.  At  the  end  of  this  time  prepare  four 
plates  from  it.  Make  each  plate  with  one  drop  from 
the  heated  bouillon  culture.  At  the  end  of  three  or  four 
days  treat  the  second  tube  in  identically  the  same  way. 
How  do  the  number  of  colonies  which  developed  from 
the  two  different  cultures  compare?  Was  there  any 
difference  in  the  time  required  for  their  development  on 
the  plates  ? 

From  a  potato  culture  of  anthrax  bacilli  which  has 
been  in  the  incubator  for  three  or  four  days,  scrape 
away  the  growth  and  carefully  break  it  up  in  10  c.c.  of 
sterilized  normal  salt  solution.  The  more  carefully  it 
is  broken  up  the  more  accurate  will  be  the  experiment. 
Place  this  in  a  bath  of  boiling  water  and  at  the  end  of 
one,  three,  five,  seven,  and  ten  minutes  make  a  plate 
upon  agar-agar  with  one  oese  of  the  contents  of  this 
tube.  Are  the  results  on  the  plates  alike  ? 


EXPEKIMENTS.  241 

Determine  the  exact  time  necessary  to  sterilize  ob- 
jects, such  as  silk  or  cotton  threads,  on  which  anthrax 
spores  have  been  dried,  by  the  steam  method  and  by  the 
hot-air  method. 

Prepare  from  the  blood  of  an  animal  just  dead  of  an- 
thrax a  bouillon  culture.  After  this  has  been  in  the 
incubator  for,  from  three  to  four  hours,  subject  it  to  a 
temperature  of  55°  C.  for  ten  minutes.  At  the  end  of 
this  time  make  plates  from  it,  and  also  inoculate  a  rabbit 
subcutaneously  with  it.  What  are  the  results?  Are 
the  colonies  on  the  plates  in  every  way  characteristic? 

Inoculate  six  Erlenmeyer  flasks  of  sterile  bouillon, 
each  containing  about  35  c  c.  of  the  medium,  from  either 
the  blood  of  an  animal  'just  dead  of  anthrax  or  from  a 
fresh  virulent  culture  in  which  no  spores  are  formed. 

Place  these  flasks  in  the  incubator  at  a  temperature 
of  42.5°  C.  At  the  end  of  five,  ten,  fifteen,  twenty, 
twenty-five,  etc.,  days  remove  a  flask.  Label  each  flask 
as  it  is  taken  from  the  incubator  with  the  exact  number 
of  days  for  which  it  had  been  at  the  temperature  of 
42.5°  C.  Study  each  flask  carefully,  both  in  its  cultural 
peculiarities  and  its  pathogenic  properties,  when  em- 
ployed on  animals. 

Are  these  cultures  identical  in  all  respects  with  those 
that  have  been  kept  at  37°  C.  ? 

If  they  differ,  in  what  respect  is  the  difference  most 
conspicuous  ? 

Should  any  of  the  animals  survive  the  inoculations 
made  from  the  different  cultures  in  the  foregoing  exper- 
iment, note  carefully  which  one  it  is,  and  after  ten  to 


242  BACTERIOLOGY. 

twelve  days  repeat  the  inoculation,  using  the  same  cul- 
ture ;  if  it  again  survives,  inoculate  it  with  the  culture 
preceding  the  one  just  used  in  the  order  of  removal  from 
the  incubator ;  if  it  still  survives,  inoculate  it  with  vir- 
ulent anthrax.  What  is  the  result  ?  How  is  the  result 
to  be  explained?  Do  the  cultures  which  were  made 
from  these  flasks  at  the  time  of  their  removal  from  the 
incubators  act  in  the  same  way  toward  animals  as  the 
organisms  growing  in  the  flasks  ?  Is  the  action  of  each 
of  these  cultures  the  same  for  mice,  guinea-pigs,  and 
rabbits  ? 

Prepare  a  2  per  cent,  solution  of  sulphuric  acid  in 
distilled  water ;  suspend  in  this  a  number  of  anthrax 
spores ;  at  the  end  of  three,  six,  and  nine  days  at  35°  C. 
inoculate  both  a  guinea-pig  and^a  rabbit.  Prepare  cul- 
tures from  this  suspension  on  the  third,  sixth,  and  ninth 
days  ;  when  the  cultures  have  developed  inoculate  a  rab- 
bit and  a  guinea-pig  from  the  culture  made  on  the  ninth 
day.  Should  the  animal  survive,  inoculate  it  again  after 
three  or  four  days  with  a  culture  made  on  the  sixth  day. 
Do  the  results  appear  in  any  way  peculiar  ? 


CHAPTER  XXIV. 

Bacteriology  of  diphtheria— Behavior  of  the  bacillus  diphtherise 
in  the  tissues  of  susceptible  animals. 

FROM  the  gray-white  deposit  on  the  fauces  of  a  diph- 
theritic patient  prepare  a  series  of  cultures  in  the  fol- 
lowing way : 

Have  at  hand  five  or  six  tubes  of  Loffler's  blood- 
serum  mixture.  (See  article  on  Media.) 

Pass  a  stout  platinum  needle,  which  has  been  steril- 
ized, into  the  membrane  and  twist  it  around  once  or 
twice  or  brush  it  gently  over  the  surface  of  the  mem- 
brane. Without  touching  it  against  anything  else  smear 
it  carefully  over  the  surface  of  one  of  the  serum 
tubes ;  without  sterilizing  it  pass  it  over  the  surface  of 
the  second,  then  the  third,  fourth,  and  fifth  tube.  Place 
these  tubes  in  the  incubator.  Then  prepare  cover-slips 
from  scrapings  from  the  membrane  on  the  fauces.  If 
the  case  is  true  diphtheria  the  tubes  will  be  ready  for 
examination  on  the  following  day. 

The  reason  that  plates  are  not  made  in  the  regular 
way  in  this  experiment  is  that  the  bacillus  of  diphtheria 
develops  much  more  luxuriantly  on  the  serum-mixture, 
from  which  plates  cannot  be  made,  than  it  does  on  the 
media  from  which  they  can  be  made.  The  method  em- 
ployed, however,  insures  a  dilution  in  the  number  of 
organisms  present,  and  this,  in  addition  to  the  fact  that 
the  bacillus  diphtherias  grows  much  more  quickly  on 


244  BACTERIOLOGY. 

the  serum  mixture  than  do  other  organisms,  makes  its 
isolation  by  this  method  a  matter  of  but  little  difficulty. 

After  twenty-four  hours  in  the  incubator  the  tubes 
will  present  a  characteristic  appearance.  Their  surfaces 
will  be  marked  here  and  there  by  more  or  less  irregular 
patches  of  a  dense  white  or  cream-colored  growth  which 
is  usually  more  dense  at  the  centre  than  at  its  irregular 
periphery. 

Except  now  and  then  when  a  few  orange-colored  col- 
onies may  be  seen,  these  large  irregular  patches  are  the 
most  conspicuous  objects  on  the  surface  of  the  serum. 
Now  and  then  almost  nothing  else  can  be  made  out  on 
the  tubes. 

The  cover-slips  made  from  the  membrane  at  the 
time  the  cultures  were  prepared  will  be  found  in 
many  cases  to  present  a  great  variety  of  organisms, 
but  conspicuous  among  them  will  be  noticed  slightly 
curved  bacilli  of  very  irregular  size  and  outline.  In 
some  cases  they  will  be  more  or  less  clubbed  at  one 
or  both  ends ;  sometimes  they  appear  spindle  in  shape, 
again  as  curved  wedges;  now  and  then  they  will  be  seen 
irregularly  segmented.  They  are  rarely  or  never  reg- 
ular in  outline.  If  the  preparation  has  been  stained 
with  Loffler's  alkaline  methylene-blue  solution  many 
of  these  irregular  rods  are  seen  to  be  marked  by  cir- 
cumscribed points  in  their  protoplasm  which  stain 
very  intensely ;  they  appear  almost  black.  This  ir- 
regularity in  outline  is  the  morphological  character- 
istic of  the  bacillus  diphtherias  of  Loffler.  It  must  be 
remembered,  however,  that  the  diagnosis  of  diphtheria 
cannot  be  made  from  the  examination  of  cover-slip 
preparations  alone,  for  there  are  other  organisms  present 
in  the  mouth  cavity,  particularly  in  the  mouth  of  those 


B.  DIPHTHERIA    ON    SERUM-MIXTURE.    245 

having  decayed  teeth,  the  morphology  of  which  is  so 
like  that  of  the  bacillus  of  diphtheria  that  they  might 
easily  be  mistaken  for  that  organism  if  subjected  to 
microscopic  examination  alone. 

The  bacillus  diphtherise  of  Loffler  (its  discoverer)  can 
readily  be  identified  by  its  cultural  peculiarities  in  con- 
nection with  its  pathogenic  activity  when  introduced 
into  tissues  of  susceptible  animals.  In  guinea-pigs  and 
kittens  the  results  of  its  growth  are  identical  with  those 
found  in  the  bodies  of  human  beings  who  have  died  of 
diphtheria. 

When  studied  in  pure  culture,  its  morphological  and 
cultural  peculiarities  are  as  follows: 

In  morphology  it  varies  greatly  in  size  and  shape, 
averaging  2.5  to  3  /"  in  length  and  0.5  to  0.8  p-  in  thick- 
ness. Its  morphological  characters  are  so  peculiar  as  te 
render  its  detection  on  cover-slip  preparations,  and  in 
sections  from  diphtheritic  membranes,  in  most  cases  an 
easy  matter.  Sometimes  appearing  as  a  regular  straight 
or  slightly  bent  rod,  with  rounded  ends,  it  is  especially 
characteristic  to  find  irregular,  bizarre  forms,  such  as  rods 
with  one  or  both  ends  swollen,  and  very  frequently  rods 
broken  at  irregular  intervals  into  short,  sharply  marked 
segments,  either  round,  oval,  or  with  straight  sides. 
Some  forms  stain  uniformly,  others  in  various  irregular 
ways,  the  most  common  being  the  appearance  of  deeply 
stained  granules  in  a  lightly  stained  bacillus. 

GROWTH  ON  SERUM-MIXTURE. — The  medium  upon 
which  it  grows  most  rapidly  and  luxuriantly,  and  which 
is  best  adapted  for  determining  its  presence  in  diphthe- 
ritic exudations  is,  as  has  been  slated,  the  blood-serum 
mixture  of  Loffler.  (See  chapter  on  Media.)  On  the 
blood-serum  mixture  the  colonies  of  the  bacillus  diph- 


246  BACTERIOLOGY. 

theriae  grow  so  much  more  rapidly  than  other  organ  isms 
usually  present  in  the  secretions  and  exudations  in  the 
throat  that  at  the  end  of  twenty-four  hours  they  are 
often  the  only  colonies  that  attract  attention,  and  if 
others  of  similar  size  are  present,  they  are  generally  of 
quite  a  different  aspect  Its  colonies  are  large,  round, 
elevated,  grayish-white,  with  a  centre  more  opaque  than 
the  slightly  irregular  periphery.  The  surface  of  the 
colony  is  at  first  moist,  but  after  a  day  or  two  rather 
dry  in  appearance. 

A  blood-serum  tube  studded  over  with  coalescent  or 
scattered  colonies  of  this  organism  is  so  characteristic 
in  appearance  that  one  can  anticipate  with  tolerable  cer- 
tainty the  results  of  microscopic  examination. 

GLYCERIN  AGAR-AGAR. — Upon  nutrient  glycerin- 
agar-agar  the  colonies  likewise  present  an  appearance 
which  may  readily  be  recognized.  They  are  in  every  way 
more  delicate  in  their  structure  than  when  on  the  serum 
mixture.  They  appear  at  first,  when  on  the  surface,  as 
very  flat,  almost  transparent,  dry,  non-glistening,  round, 
points  which  are  not  elevated  above  the  surface  upon 
which  they  are  growing.  When  slightly  magnified 
they  are  seen  to  be  granular,  and  present  an  irregular 
central  marking  which  is  more  dense  and  darker  by 
transmitted  light  than  the  thin,  delicate  zone  which  sur- 
rounds it.  The  periphery  of  the  colony  is  marked  by  an 
irregularly  notched  appearance.  These  colonies  are  al- 
ways quite  dry  in  appearance.  When  deep  down  in  the 
agar-agar  they  are  coarsely  granular.  They  rarely  exceed 
3  mm.  in  diameter. 

GELATIN. — On  gelatin  the  colonies  develop  much 
more  slowly  than  on  the  other  media  which  can  be 
retained  at  a  higher  temperature.  They  rarely  present 


B.    DIPHTHERIA    ON    POTATO.  247 

their  characteristic  appearances  on  gelatin  in  less  than 
seventy-two  hours. 

They  then  appear  as  flat,  dry,  translucent  points, 
usually  round  in  outline. 

When  magnified  slightly,  the  centre  is  seen  to  be 
more  dense  than  the  surrounding  zone  or  zones,  for  they 
are  sometimes  marked  by  a  concentric  arrangement  of 
zones.  The  periphery  is  irregularly  notched.  Like  the 
colonies  seen  on  agar-agar,  they  are  granular,  but  are 
much  more  granular  when  seen  in  the  depths  of  the 
gelatin  than  when  on  its  surface.  On  gelatin  the  colonies 
rarely  become  very  large;  usually  they  do  not  reach  a 
diameter  of  over  1.5  mm. 

BOUILLON. — In  bouillon  it  usually  grows  in  fine 
clumps,  which  fall  to  the  bottom  of  the  tube,  or  become 
deposited  on  its  sides  without  causing  a  diffuse  clouding 
of  the  bouillon.  There  are  sometimes  exceptions  to  this 
naked-eye  appearance.  The  bouillon  may  appear  diffusely 
clouded,  but  if  one  inspects  it  very  closely,  particularly 
if  one  examines  it  microscopically  in  the  form  of  a 
hanging  drop,  the  arrangement  in  clumps  will  still  be 
seen,  but  they  are  so  small  as  not  to  have  been  detected 
by  the  unaided  eye. 

In  bouillon  which  is  kept  at  a  temperature  of 
35°-37°  C.  for  a  long  time,  a  soft,  whitish  membrane 
often  forms  over  a  part  of  the  surface. 

Changes  in  Reactions  of  the  Bouillon. — The  reaction 
of  the  bouillon  becomes  at  first  acid,  and,  subsequently, 
again  alkaline,  changes  which  can  be  well  observed  in 
cultivations  in  bouillon  to  which  a  little  rosolic  acid 
has  been  added. 

POTATO.— On  potato  at  a  temperature  of  35°-37°  C. 
its  growth  after  several  days  is  entirely  invisible ;  only 


248  BACTERIOLOGY. 

a  thin,  dry  glaze  can  be  noticed  at  the  point  at  which  the 
potato  was  inoculated.  Microscopic  examination  of  the 
potato  after  twenty-four  hours  at  35°-37°  C.  shows  a 
decided  increase  in  the  number  of  individual  organisms 
planted. 

STAB  AND  SLANT  CULTURES. — In  stab  and  slant 
cultures  on  both  gelatin  and  glycerin  agar-agar,  the 
surface  growth  is  seen  to  predominate  over  that  along 
the  track  of  the  needle  in  the  depths  of  the  media. 

Isolated  colonies  on  the  surface  of  either  of  the  media 
in  this  method  of  cultivation  present  the  same  charac- 
teristics that  have  been  given  for  the  colonies. 

The  growth  in  simple  stab  cultures  does  not  extend 
laterally  very  far  beyond  the  point  at  which  the  needle 
entered  the  medium. 

It  is  a  non-motile  organism. 

It  does  not  form  spores. 

It  is  killed  in  ten  minutes  by  a  temperature  of 
58°  C. 

It  grows  at  temperatures  ranging  from  22°  C.  to 
37°  C.,  but  most  luxuriantly  at  the  latter  temperature. 

Its  growth  in  the  presence  of  oxygen  is  more  active 
than  when  the  gas  is  excluded. 

STAINING. — In  cover-slip  preparations  made  either 
from  the  fauces  of  a  diphtheritic  patient,  or  from  a  pure 
culture  of  the  organism,  it  is  seen  to  stain  readily  with 
the  ordinary  aniline  dyes.  It  stains  also  by  the  method 
of  Gram,  but  the  best  results  are  those  obtained  by  the 
use  of  Loffler's  alkaline  methylene-blue  solution ;  this 
brings  out  the  dark  points  in  the  protoplasmic  body  of 
the  bacilli  and  aids  thus  in  their  identification. 

For  the  purpose  of  demonstrating  the  Loffler  bacilli 
in  sections  of  diphtheritic  membrane,  both  the  Gram 


INOCULATIONS    OF    B.   DIPHTHERIA .      249 

method  and  the  fibrin  method  of  Weigert  give  excellent 
results. 

PATHOGENIC  PROPERTIES. — When  inoculated  sub- 
cutaneously  into  the  bodies  of  susceptible  animals  the 
result  is  not  the  production  of  a  septicaemia  as  is  seen 
to  follow  the  introduction  into  animals  of  certain  other 
organisms  with  which  we  shall  have  to  deal.  The 
bacillus  of  diphtheria  remains  localized  at  the  point 
of  inoculation,  never  spreading  further  than  the  nearest 
lymphatic  glands.  It  develops  at  the  point  in  the  tissues 
at  which  it  is  deposited,  and  during  its  development  gives 
rise  to  changes  in  the  tissues  which  result  entirely  from 
the  absorption  into  the  circulation  of  poisonous  albumins 
produced  by  the  bacilli  in  the  course  of  their  develop- 
ment. 

If  a  very  minute  portion  of  either  a  solid  or  fluid 
pure  culture  of  this  organism  is  introduced  into  the 
subcutaneous  tissues  of  a  guinea-pig  or  kitten,  death  of 
the  animal  is  seen  to  ensue  in  from  twenty-four  hours 
to  five  days.  The  usual  changes  are  an  extensive  local 
oadema  with  more  or  less  hypersemia  and  ecchymosis  at 
the  site  of  inoculation ;  frequently  swollen  and  reddened 
lymphatic  glands ;  increased  serous  fluid  in  the  perito- 
neum, pleura,  and  pericardium ;  enlarged  and  hemor- 
rhagic  supra-renal  capsules ;  occasionally  slightly  swollen 
spleen  ;  sometimes  fatty  degeneration  in  the  liver,  kidney, 
and  myocardium.  The  bacilli  are  always  to  be  found 
at  the  seat  of  inoculation,  most  abundant  in  the  grayish- 
white  fibrino-purulent  exudate  present  at  the  point  of 
inoculation,  and  becoming  fewer  at  a  distance  from  this, 
so  that  the  more  remote  parts  of  the  O3dematous  fluid  do 
not  contain  any  bacilli.  The  bacilli  are  found  not  only 
free,  but  contained  in  large  number  in  leucocytes,  some 


250  BACTERIOLOGY. 

of  which  have  fragmented  nuclei  or  have  lost  their  nuclei. 
The  bacilli  within  the  leucocytes  as  well  as  some  outside 
frequently  stain  very  faintly  and  irregularly,  and  may 
appear  disintegrated  and  dead. 

In  all  cases  culture-tubes  inoculated  with  the  blood, 
spleen,  liver,  kidneys,  supra-renal  capsules,  distant 
lymphatic  glands,  and  serous  transudates  yield  nega- 
tive growths.  Negative  results  are  also  always  obtained 
when  these  organs  are  examined  microscopically  for  the 
bacilli. 

Microscopic  examinations  of  the  tissues  about  the  seat 
of  inoculation,  as  well  as  of  the  liver,  spleen,  kidneys, 
lymphatic  glands,  and  elsewhere,  show  a  condition  of 
extensive  cell-death  which  is  characterized  by  an  extreme 
degree  of  fragmentation  of  the  nuclei  of  the  cells  of  these 
parts.  These  peculiar  alterations,  as  Oertel  has  shown, 
in  their  distribution  are  characteristic  of  human  diph- 
theria, and  the  demonstration  of  similar  changes  in 
animals  inoculated  with  this  organism  is  no  small 
additional  proof  that  diphtheria  is  caused  by  it. 

An  affection  may  be  produced  by  the  inoculation  of 
certain  animals  in  all  respects  identical  with  the  disease 
diphtheria  as  it  exists  in  man.  If  one  opens  the  trachea 
of  a  kitten  and  rubs  upon  the  mucous  membrane  a  small 
portion  of  a  pure  culture  of  this  organism,  the  death  of 
the  animal  will  ensue  in  from  two  to  four  days.  At 
autopsy  the  wound  will  be  found  covered  with  a  gray- 
ish, adherent,  necrotic,  distinctly  diphtheritic  layer. 
Around  the  wound  the  subcutaneous  tissues  will  be 
oedematous.  The  lymphatic  glands  at  the  angle  of  the 
jaws  will  be  swollen  and  reddened.  The  mucous  mem- 
brane of  the  trachea  at  the  point  upon  which  the  bacilli 
were  deposited  will  be  covered  with  a  tolerably  firm, 


INOCULATIONS    OF    B.   DIPHTHEKI^E.       251 

grayish-white,  loosely  attached  pseudo-membrane  in  all 
respects  identical  with  the  croupous  membrane  observed 
in  the  same  situation  in  cases  of  human  diphtheria. 
In  the  pseudo-membrane  and  in  the  oedematous  fluid 
about  the  skin-wound,  bacilli  diphtherias  may  be  found 
both  in  cover-slips  and  in  cultures. 

From  what  we  have  seen — the  localization  of  the  bacilli 
at  the  point  of  inoculation,  their  absence  from  the  internal 
organs,  and  the  changes  brought  about  in  the  cellular 
elements  of  the  internal  organs — there  is  but  one  inter- 
pretation for  this  process,  and  that  is  the  production  of 
a  soluble  poison  by  the  bacteria  growing  at  the  seat  of 
inoculation,  which  gains  access  to  the  circulation  and 
produces  the  changes  that  we  observe  in  the  tissues  of 
the  internal  viscera. 

This  poison  has  been  isolated  from  cultures  of  the 
bacillus  of  diphtheria  by  Brieger  and  Frankel.  It  is 
found  to  belong,  not  to  the  crystallizable  ptomaines,  but 
to  the  toxic  albumins.  By  the  introduction  of  this  tox- 
albumin,  as  it  is  called,  into  the  tissues  of  guinea-pigs 
and  rabbits  the  same  pathological  alterations  may  be 
produced  that  we  have  seen  to  follow  the  result  of  inocu- 
lation with  the  bacilli  themselves,  except,  perhaps,  the 
production  of  false  membrane. 

Prepare  cover-slip  preparations  from  the  mouth  cavity 
of  healthy  individuals  and  from  those  having  decayed 
teeth.  Do  they  correspond  in  any  way  with  those  made 
from  diphtheria  ?  Do  the  same  with  different  forms  of 
sore-throat.  Do  the  peculiarities  of  any  of  the  organ- 
isms suggest  those  of  the  bacillus  of  diphtheria?  Wherein 
is  the  difference? 

In  cultures  and  cover-slips  made  from  both  diphtheria 


252  BACTERIOLOGY. 

and  innocent  sore-throats  are  there  any  organisms  which 
are  almost  constantly  present?  Which  are  they,  and 
what  are  their  characteristics  ? 

In  the  anginas  of  scarlet  fever  which  are  the  pre- 
dominating organisms? 

In  their  cultural  and  morphological  peculiarities,  do 
these  organisms  simulate  any  of  the  different  species 
with  which  you  have  been  working? 


CHAPTER    XXV. 

Experiments  illustrating  precautions  to  be  taken  in  the  study  of 
disinfectants  and  antiseptics — Skin  disinfection. 

INTO  each  of  three  tubes  containing  10  c.c. — one 
of  normal  salt  solution,  another  of  bouillon,  a  third  of 
fluid  blood-serum — add  as  much  of  a  culture  of  the 
staphylococcus  pyogenes  aureus  as  can  be  held  upon 
the  looped  platinum  needle.  Mix  this  thoroughly,  so 
that  no  clumps  exist,  and  then  add  exactly  10  c.c.  of 
1 : 500  solution  of  corrosive  sublimate.  Mix  it  thor- 
oughly, and  at  the  end  of  three  minutes  transfer  a  drop 
from  each  tube  into  a  tube  of  liquefied  agar-agar,  and 
pour  this  into  a  Petri  dish.  Label  each  dish  carefully 
and  place  them  in  the  incubator.  Are  the  results  the 
same  in  all  the  plates?  How  are  the  differences  to  be 
explained  ? 

Into  each  of  two  tubes  containing  10  c.c. — the  one  of 
normal  salt  solution,  the  other  of  bouillon — add  as  much 
of  a  spore-containing  culture  of  anthrax  bacilli  as  can 
be  held  upon  the  loop  of  the  platinum  wire.  Mix  this 
thoroughly  so  that  no  clumps  exist  and  then  add  ex- 
actly 10  c.c.  of  a  1:500  solution  of  corrosive  subli- 
mate. Mix  thoroughly  and  at  the  end  of  five  minutes 
transfer  a  drop  from  each  tube  into  a  tube  of  liquefied 
agar-agar.  Pour  this  immediately  into  a  Petri  dish. 
Label  each  dish  carefully  and  place  them  in  the  incuba- 
tor. Note  the  results  at  the  end  of  twenty-four,  forty- 
eight,  and  seventy-two  hours.  How  do  you  explain  them  ? 

12 


254  BACTERIOLOGY. 

Make  identically  the  same  experiment  with  a  spore- 
containing  culture  of  anthrax,  except  that  the  drop  from 
the  mixture  will  be  transferred  to  10  c.c.  of  a  mixture 
of  equal  parts  of  ammonium  sulphide  and  sterilized  dis- 
tilled water.  After  remaining  in  this  for  about  half  a 
minute,  a  drop  will  be  transferred  to  a  tube  of  lique- 
fied agar-agar,  poured  into  Petri  dishes,  label  UK!,  and 
placed  in  the  incubator.  Note  the  results.  How  are 
they  explained  ? 

Prepare  a  1  : 1000  solution  of  corrosive  sublimate. 
To  each  of  twelve  bouillon  tubes  containing  exactly  10 
c.c.  add :  one  drop  to  the  first  one,  two  drops  to  the 
second,  and  so  on  until  the  last  tube  has  had  twelve  drops 
added  to  it.  Mix  thoroughly  and  then  inoculate  each 
with  one  wire  loopful  of  a  bouillon  culture  of  staphylo- 
coccus  pyogenes  aureus.  Place  them  all  in  the  incubator 
after  carefully  labelling  them.  Note  the  order  in  which 
growth  appears. 

Do  the  same  with  anthrax  spores,  with  spores  of  bacillus 
subtilis,  with  the  typhoid  bacillus,  and  see  how  the  re- 
sults compare.  From  these  experiments  what  will  be 
the  strength  of  corrosive  sublimate  necessary  to  act  as 
an  antiseptic  under  these  conditions  for  the  organisms 
employed  ? 

Make  a  similar  series  of  experiments,  using  a  five  per 
cent,  solution  of  carbolic  acid. 

Determine  the  antiseptic  point  in  bouillon  of  the 
common  disinfectants  for  the  organisms  with  which  you 
are  working. 

Determine  the  time  necessary  for  the  destruction  of 
the  organisms  with  which  you  are  working,  by  corro- 


EXPERIMENTS.  255 

sive  sublimate  in  1  : 1000  solution,  under  different  con- 
ditions— with  and  without  the  presence  of  albuminous 
bodies  other  than  the  bacteria  and  under  varying  condi- 
tions of  temperature. 

In  making  these  experiments  be  careful  to  guard 
against  the  introduction  of  enough  sublimate  into  the 
agar-agar  from  which  the  Petri  plate  is  to  be  made  to 
inhibit  the  growth  of  the  organisms  which  may  not  have 
been  destroyed  by  the  sublimate.  This  may  be  done  by 
transferring  two  drops  from  the  mixture  of  sublimate 
and  organisms  into  not  less  than  10  c.c.  of  sterilized  salt 
solution  in  which  they  may  be  thoroughly  shaken  for 
from  one  to  two  minutes,  or  into  the  solution  of  am- 
monium sulphide  of  the  strength  given. 

To  10  c.c.  of  a  bouillon  culture  of  staphylococcus 
pyogenes  aureus,  or  anthrax  spores,  add  10  c.c.  of  corro- 
sive sublimate  in  1  : 500  solution,  and  allow  it  to  re- 
main in  contact  with  the  organisms  for  only  one-half  the 
time  necessary  to  destroy  them  (use  an  organism  for 
which  this  has  been  determined  under  these  conditions). 
Then  transfer  a  drop  of  the  mixture  to  each  of  three 
liquefied  agar-agar  tubes  and  pour  them  into  Petri 
dishes.  Place  them  in  the  incubator  and  observe  them 
for  twenty-four,  forty-eight,  and  seventy-two  hours. 
No  growth  occurs.  How  is  this  to  be  accounted  for? 

At  the  end  of  seventy-two  hours  inoculate  all  of  these 
plates  with  a  culture  of  the  same  organism  which  has 
not  been  exposed  to  sublimate,  by  taking  up  bits  of  the 
culture  on  the  needle  and  drawing  it  across  the  plates. 
A  growth  now  results.  We  have  here  an  experiment  in 
which  organisms  which  have  been  exposed  to  sublimate 
for  a  much  shorter  time  than  is  necessary  to  destroy 


256  BACTERIOLOGY. 

them,  when  transferred  directly  to  a  culture  medium  do 
not  grow,  and  yet,  when  the  same  organism  which  lias 
not  been  exposed  to  sublimate  is  planted  upon  the  same 
medium  it  does  grow.  How  is  this  to  be  accounted  for  ? 

SKIN-DISINFECTION. — With  a  sterilized  knife  scrape 
from  the  skin  of  the  hands,  at  the  root  of  the  nails  and 
under  the  nails,  small  particles  of  epidermis.  Prepare 
plates  from  them.  Note  the  results. 

Wash  the  hands  carefully  for  ten  minutes  in  hot 
water  and  scrub  them  during  this  time1  witli  soap  and  a 
sterilized  brush.  Rinse  them  in  hot  water.  Again  prepare 
plates  from  scrapings  of  the  skin  on  the  fingers,  at  the 
root  of  the  nails,  and  under  the  nails.  Note  the  results. 

Again  wash  as  before  in  hot  water  with  soap  and 
brush,  rinse  in  hot  water,  then  soak  the  hands  for  five 
minutes  in  1  : 1000  corrosive  sublimate  solution,  and,  as 
before,  prepare  plates  from  scrapings  from  the  same 
localities.  Note  the  results. 

Repeat  this  latter  procedure  in  exactly  the  same  way, 
but  before  taking  the  scrapings  let  someone  pour  am- 
monium sulphide  over  the  points  from  which  the  scrap- 
ings are  to  be  made.  After  it  has  been  on  the  hands 
about  three  minutes  again  scrape  and  note  the  results 
upon  plates  made  from  the  scrapings. 

Wash  as  before  in  hot  water  and  soap,  rinse  iu  clean 
hot  water,  immerse  for  a  minute  or  two  in  alcohol,  after 
this  in  1 : 1000  sublimate  solution,  and  finally  in  am- 
monium sulphide,  and  then  prepare  plates  from  scrapings 
from  the  points  mentioned. 

In  what  way  do  the  results  of  these  experiments  differ 
the  one  from  the  other  ? 

To  what  are  these  differences  due  ? 

What  have  these  experiments  taught  ? 


SKIN-DISINFECTION.  257 

In  making  the  above  experiments  it  must  be  remem- 
bered that  the  strictest  eare  is  necessary  in  order  to  pre- 
vent the  access  of  germs  from  without  into  our  media. 
The  hand  upon  which  the  experiment  is  being  per- 
formed must  be  held  away  from  the  body  and  must  not 
touch  any  object  not  concerned  in  the  experiment.  The 
scraping  should  be  done  with  the  point  of  a  knife  which 
has  been  sterilized  in  the  flame  and  allowed  to  cool 
down.  The  scrapings  may  be  transferred  directly  from 
the  knife-point  to  the  gelatin  by  means  of  a  sterilized 
platinum  wire  loop. 

The  brush  used  should  be  thoroughly  cleansed  and 
always  kept  in  1  : 1000  solution  of  corrosive  sublimate. 
It  should  be  washed  in  hot  water  before  using. 


INDEX. 


ABSCESSES,  miliary,  221 

x\     microscopic  appearances  of, 

222,  223 

Aerobic  organisms,  28 
Agar-agar,  57,  65 

clarification  of,  65 
filtration  of,  66 
peculiarities  of,  57,  58 
Air,  bacteriological  analysis  of,  186 
Petri's  method  for,  187 
Sedg wick's  method  for, 

187-191 
Anaerobic  organisms,  27,  28 

methods  of  cultivation,  116- 

119 

method  of  Buchner,  117 
Esmarch,  119 
Frankel,  118 
Hesse,  117 

Kitasato  and  Weil,  119 
Koch,  117 
Liborius,  119 

Aniline  dyes  as  aids  in  differenti- 
ation, 115 

Animals,  inoculation  of,  151-158 
intra-venous,  154-158 
instruments  for,  156-158 
subcutaneous,  151 
post-mortem  examination  of, 

159-163 

cultures  from  tissues,  161 
disinfection    of    imple- 
ments, 163 

disposal  of  remains,  163 
external  inspection,  159 
incision  through  skin . 

159 

opening  the  cavities,  160 
position  of  the  animal, 
159 


Anthrax,  232 

bacillus  of,  232-238 

cultivation  of,  234-236 
experiments  with,  238- 

242 
inoculation    with,    236- 

238 

staining  of,  236 
Antiseptics,  53 

experiments  upon,  254 
Apparatus  for  air  analysis,  Petri's, 

187 

Sedgwick's  188 
for    counting    colonies,    Es- 

march's,  184 
Wolffhiigel's,  182 
Autoclav  or  digestor,  46 


BACILLI,  29-32 
Bacillus  subtilis,  173 
of  tuberculosis,  211 
Bacteria,     capsule     surrounding, 

classification  of,  29 
constant    characteristics    of, 

104 

definition  of,  22 
flagellse  upon,  136  ^ 
isolation  of,  on  solid  media, 

54 
Koch's  observation,  54- 

57 
microscopic  examination  of, 

105 
morphology  of,  29 

constancy  of,  31 
motility  of,  36 
multiplication  of,  32,  33 
nutrition  of,  25-27 


2  GO 


INDEX. 


r>;u-U'ria,  reactions  produced  by, 

111 

relation  to  oxygen,  27.  •> 
to  staining  reagents,  115 
to  temperature,  28 
role  in  nature,  23-25 
-v^tematic  study  of,  104-121 

scheme  for^  1(54,  165 
Bacterium  coli  commune,  230,  231 
I  Jen  eh  es,  glass,  for  holding  plates, 

86 
Blood-serum,  69 

Loffler's  mixture,  75,  7<> 
preparation  of,  69 
preservation  of,  73,  74 
solidification  of,  71-73 
sterilisation  of,  71 
Bonnet,  20 

Bottles  for  staining  solutions,  128 
Bouillon,  57-59 

neutralisation  of,  (iO,  61 
Box,  iron,  in  which  to  sterilize 

plates,  84 

Brownian  motion,  110 
Bulb  for  water  samples,  17'.' 
Burner,  Koch's  safety,  93,  94 
rose  or  crown,  47 


/  1 1 1  L<  >R<  )PHYLL,      definition 
\J     of,  22 
Cohn,  20 

Colonies,   characteristic    appear- 
ance of,  168,  169 
study  of,  99-101 
Cooling-stage,  84 
Cover-slips,  cleaning  of,  12.". 
impression,  126 
microscopic  examination  of, 

107 

preparation  of,  123-127 
>U'|is  in  making,  124 
Culture-dish  for  plate-.  >~> 
Cultures,  gelatin,  113 
hanging-drop,  109 
potato,  113 

in  tube,  69 
pure,  101 
>tab  and  smear,  101-103 

method  of  making,  101, 
102 


]\EC<  >L<  >K1Z1  N<  i  snlutions,139 
1^     Decomposition,  22,  23 
Diphtheria,  2-13 

bacillus  of,  245  252 

cultivation  of,  245-248 
pathogenic  properties  of, 

249-252 
preparation  of  cultmv> 

from,  243-245 
staining  of,  248 
Diplococci,  30 

Diplococcus  of  pneumonia,  194 
Disinfectants,  experiments  upon, 

263-257 
Disinfection,  49-53 

agents  employed  for,  52,  53 
in  the  laboratory,  52 
inorganic  salts  for,  49-51 
methods  of,  52 


ERYSIPELAS,  224 
Esmarch's  roll  tubes,  87 
modification  of  Esmarch's 

method,  88 
ad  vantages  of  this  pro- 
cess, 89 

roll  tubes  of  agar-agar,  S9 
tube  being  rolled  on  ice,  88 
Experiments,  167 

exposure  and    contact,   169, 
170 

r\V(VLTATlVE    aerobic   and 
F      anaerobic  oinani-ms,  28 
Fermentation,  22,  23,  116 
Filters,  preparation  of,  64    ' 
Flagellae,     Loffler's     method    of 

staining,  136 

Flasks,  etc.,  cleaning  of,  77 
Funnel     for    filling    Sedgwick's 

aerobioscope,  190 
for  filling  test-tubes,  etc.,  79 


pELATIN,  57-62 
U    clarification  of,  63 

filtration  of,  63 

peculiarities  of,  57,  •"» 
<  niarniari's  agar-gclatin,  76 


INDEX. 


261 


HANGING  -  DROP      prepara- 
tions, 108 
Herile,  17 
Hoffmann,  19 
Hypodermatic  needles,  156 
syringes,  158 


INCUBATORS,  91-93 

1  Indol,  its  production   by  bac- 
teria, 120 

method  for  detecting  its  pres- 
ence, 120,  121 

Introduction,  13 


I  EEUWENHOEK'S  discover- 
L    ies,  13-15 

Lens  for  counting  colonies,  183 
Levelling  tripod,  84 
Loffler's    blood-serum    mixture, 
75,76 


M 


EDIA,  59-76 
agar-agar,  65-67 

clarification  of,  65,  66 

filtration  of,  66 

solution  of,  65,  66 
blood-serum,  69-74 

precautions    in    obtain- 
ing, 69,  70 

preservation  of,  73,  74 

solidifying,  71-73 

sterilization  of,  71 
bouillon,  59 

neutralization  of,  59-61 
gelatin,  62-65 

clarification  of,  63,  64 

filtration  of,  63 

neutralization  of,  62 
meat  infusion,  76 
potatoes,  67-69 

for  test-tube  cultures,  68 
special,  74-76 

agar -gelatin  of  Guarni- 
ari,  76 

blood-serum  mixture  of 
Loffler,  75 

Dunham's  peptone  solu- 
tion, 75 


Media,    special :    Dunham's  pep- 
tone solution,  with  ro- 
solic  acid,  75 
milk,  74 

as  a  solid  medium, 

74,  75 

with  litmus,  74 
Micrococci,  29 

Micrococcus  of  sputum  septicae- 
mia, 194-197 
tetragenus,  197 

animals    susceptible   to, 

200 

description  of,  198-200 
results     of    inoculation 

with,  197 
Microscope,  105-107 

adjustment,  coarse,  106 

fine,  106 

condenser,  sub-stage,  106 
immersion  system,  oil,  106 

steps  in  using,  107 
objective,  105 
ocular,  105 
reflector,  106 
stage,  106 
Microtome,  142 
Mouse-holder,  152,  153 

YfEEDHAM,  18 

li     Nitrites,  121 

ifc  AESE,"  platinum,  82 

V  Oil-immersion  system,  steps 

in  using,  107 
Ozanam,  17 


PARASITES,  22 
I     Pasteur,  17-19 
Pasteur  and  Chevreul,  19 
Peptone  solution,  Dunham's,  75 

-rosolic-acid  solution,  75 
Petri's  dish,  87 

modification  of  Koch's  meth- 
od, 86 

Plates,  technique  of  making,  81 
Platinum  needles,  loops  ("oese"), 

81,582 
Plenciz,  16 


262 


INDEX. 


Piu'iimococcus  of  Frankel,  194 
Potatoes,  invpa ration  of,  67 

for  test-tube  cultures,  68,  69 
sterilization  of,  69 
Practical  application  of  bacterio- 
logical methods,  167 
materials    for    starting, 

167 
Pus  organisms,  219-224 


1 )  EGULATOK,  gas-pressure,  97, 
thermo-,  94-97 


C  APKOPHYTES,  definition  of, 
O    22 
Sarciniae,  30 
Schroder  and  Dusch,  19 
Schulz,  19 
Schwann,  19 
Section-cutting,  141 
Septicaemia,  definition  of,  194 
sputum,  194 

organism  concerned  in, 

195 
its  different  names, 

194 
pathological  alterations 

in,  194,  195 
Skin  -  disinfection,     experiments 

upon,  255-257 

Soil,  bacteriological  study  of,  191 
Sjiallanzani,  18 
Spirilli,  28-32 
Spore-formation,  33-36 
study  of,  110 

method  for  the,  111-113 
Spores,  staining  of,  134 

Moeller's  method,  l.°,o 
Sputum,  tubercular,  192 

microscopic  examination  of, 

192 
results  of  inoculation  with, 

194-2K) 
septicaemia  of  micrococ- 

cus  tetragenus,  197 
sputum  septicaemia,  194 
tuberculosis,  201 
Staining  in  general,  138 
methods  of,  122-150 


Staining  solutions  employed,  127 

bottles  for,  1 2S  ' 
special  solutions,  12'.) -1 :'.:'. 
acetic  acid  method,  133 
Gram's  stain,  133 
Koch-Ehrlich,  129 
Loffler's  blue,  129 
Ziehl's    carbol    fuch>'m. 
130 

Staphylococci,  29 

Staphylococcus    pyogenes   allm>, 

aureus,  217 
citreus,  224 
Sterilization,  37 

apparatus  employed  in,  43-47 
dry  heat,  37 

its  applications,  38 
experiments  in,  171-175 
hot-air  method,  174 
precautions,  171 
steam  method,  171-174 
temperature    in     steril- 
izer, 171 
methods  of,  39 

intermittent,  at  high  ten i - 

perature,  40,  41 
at  low  temperature, 

41,42 

under  pressure,  42 
steam,  38 

its  application,  39 
Sterilizer,  blood-serum,  72 
hot-air,  47 
steam,  Arnold's,  45 

Koch's,  43 
Streptococci,  29 
Streptococcus  pyogenes,  224 
Suppuration,  217 

organisms  concerned  in  am  t » •. 

219 
cultural  peculiarities  of, 

217-219 

results     of    inoculation 
with,  219-223 


TEST-TUBE  cleaner,  77 
1  Test -tubes,  cleaning  of,  77 
filling  for  Esmarch  tubes,  80 
with  media,  78,  79 


I N  D  E  X . 


263 


Test-tubes,  plugging  with  cotton, 

78 

position  after  filling,  80 
sterilization  after  tilling,  79 
sterilization  of,  78 
Tetrads,  30 

Tissues,  cultures  from,  161 
hardening  of,  141 
imbedding,  142 
staining  of  bacteria  in,  140- 

143 

steps  in  the  process,  145 
special  methods  of  staining, 

146 

dahlia  method,  147 
dry  method,  150 
Erhlich's  method,  150 
Gram's  method,  146 
Kiihne's  method,  148 
Weigert's  method,  148 
Ziehl-Neelsen,  150 
Tripod  for  levelling  plates,  83 
Tuberculosis,  201 
bacillus  of,  211 

cultivation  from  the  tis- 
sues, 212 
cultural  peculiarities  of, 

213 

methods  of  staining,  131 
NuttalPs    modifica- 
tion, 132 
dry  method,  150 
Erhlich,  150 
Ziehl-Neelsen,  150 
microscopic  appearance 

of,  214 
staining  peculiarities  of, 

215 

cavity -formation,  206 
diffuse  caseation,  205 
encapsulation  of  tubercular 
foci,  206 


Tuberculosis,  infection,  modes  of, 

207 

primary,  206 

location  of  the  bacilli  in,  209 
manifestations  of,  201-203 
miliary  tubercles,  203 
susceptibility  of  animals  to, 

216 
Tyndall,  20 
Typhoid  fever,  225 

bacillus  of,  225-227 
in  tissues,  227,  228 
results    of    inoculation 
with,  228-230 


WATEK,  176 
general    observations    upon 
bacteriological  analysis  of, 
185 
qualitative      bacteriological 

analysis  of,  176 
precautions  in  obtaining 

samples  for,  177 
preliminary  steps  in,  177 
quantitative    bacteriological 

analysis  of,  178 
counting  the  colonies  in, 

181 
apparatus  for,  182, 

183 
dilution    of   water  for, 

180,  181 

obtaining  sample  for,  179 
preliminary  steps  in,  180 
source  of  error  in,  1 85 
relation  to  epidemics,  176 
typhoid  organisms  in,  230 


700GL(EA,  31 

L 


XB  65622 


010L-OGJ 

G 

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